Vascular endothelial growth factor-2

ABSTRACT

The present invention is directed to VEGF-2 polynucleotides and polypeptides and methods of using such polynucleotides and polypeptides. In particular, provided are methods of treating retinal disorders with VEGF-2 polynucleotides and polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/499,468, filed Feb. 7, 2000, which claims benefit of U.S. ProvisionalApplication No. 60/119,179, filed on Feb. 8, 1999, U.S. ProvisionalApplication No. 60/119,926, filed Feb. 12, 1999, U.S. ProvisionalApplication No. 60/137,796, filed Jun. 3, 1999, and U.S. ProvisionalApplication No. 60/171,505, filed Dec. 22, 1999. Each of the fouraforementioned applications are hereby incorporated by reference intheir entireties.

REFERENCE TO SEQUENCE LISTING ON COMPACT DISC

This application refers to a “Sequence Listing” listed below, which isprovided as an electronic document on two identical compact discs(CD-R), labeled “Copy 1” and “Copy 2.” These compact discs each containthe file “PF112U1D1 SeqList.txt” (created Feb. 25, 2005, bytesize=52,861 bytes), which is hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. The polypeptides of the presentinvention have been identified as members of the vascular endothelialgrowth factor family. More particularly, the polypeptides of the presentinvention are human vascular endothelial growth factor 2 (VEGF2). Theinvention also relates to inhibiting the action of such polypeptides.Additionally, the present invention relates to antibodies directed tothe polypeptides of the present invention. The present invention alsorelates to the administration of vascular endothelial growth factor 2(VEGF-2) polynucleotides and polypeptides to treat disorders of orinjuries to photoreceptor cells.

2. Related Art

The formation of new blood vessels, or angiogenesis, is essential forembryonic development, subsequent growth, and tissue repair.Angiogenesis is also an essential part of certain pathologicalconditions, such as neoplasia (i.e., tumors and gliomas). Abnormalangiogenesis is associated with other diseases such as inflammation,rheumatoid arthritis, psoriasis, and diabetic retinopathy (Folkman, J.and Klagsbrun, M., Science 235:442-447(1987)).

Both acidic and basic fibroblast growth factor molecules are mitogensfor endothelial cells and other cell types. Angiotropin and angiogenincan induce angiogenesis, although their functions are unclear (Folkman,J., Cancer Medicine, Lea and Febiger Press, pp. 153-170 (1993)). Ahighly selective mitogen for vascular endothelial cells is vascularendothelial growth factor or VEGF (Ferrara, N. et al., Endocr. Rev.13:19-32 (1992)), which is also known as vascular permeability factor(VPF).

Vascular endothelial growth factor is a secreted angiogenic mitogenwhose target cell specificity appears to be restricted to vascularendothelial cells. The murine VEGF gene has been characterized and itsexpression pattern in embryogenesis has been analyzed. A persistentexpression of VEGF was observed in epithelial cells adjacent tofenestrated endothelium, e.g., in choroid plexus and kidney glomeruli.The data was consistent with a role of VEGF as a multifunctionalregulator of endothelial cell growth and differentiation (Breier, G. etal., Development 114:521-532 (1992)).

VEGF shares sequence homology with human platelet-derived growthfactors, PDGFa and PDGFb (Leung, D. W., et al., Science 246:1306-1309,(1989)). The extent of homology is about 21% and 23%, respectively.Eight cysteine residues contributing to disulfide-bond formation arestrictly conserved in these proteins. Although they are similar, thereare specific differences between VEGF and PDGF. While PDGF is a majorgrowth factor for connective tissue, VEGF is highly specific forendothelial cells. Alternatively spliced mRNAs have been identified forboth VEGF, PLGF, and PDGF and these different splicing products differin biological activity and in receptor-binding specificity. VEGF andPDGF function as homo-dimers or hetero-dimers and bind to receptorswhich elicit intrinsic tyrosine kinase activity following receptordimerization.

VEGF has four different forms of 121, 165, 189 and 206 amino acids dueto alternative splicing. VEGF121 and VEGF165 are soluble and are capableof promoting angiogenesis, whereas VEGF189 and VEGF206 are bound toheparin containing proteoglycans in the cell surface. The temporal andspatial expression of VEGF has been correlated with physiologicalproliferation of the blood vessels (Gajdusek, C. M., and Carbon, S. J.,Cell Physiol.139:570-579 (1989); McNeil, P. L., et al., J. Cell. Biol.109:811-822 (1989)). Its high affinity binding sites are localized onlyon endothelial cells in tissue sections (Jakeman, L. B., et al., Clin.Invest. 89:244-253 (1989)). The factor can be isolated from pituitarycells and several tumor cell lines, and has been implicated in somehuman gliomas (Plate, K. H., Nature 359:845-848 (1992)). Interestingly,expression of VEGF121 or VEGF165 confers on Chinese hamster ovary cellsthe ability to form tumors in nude mice (Ferrara, N. et al., J. Clin.Invest. 91:160-170 (1993)). The inhibition of VEGF function by anti-VEGFmonoclonal antibodies was shown to inhibit tumor growth inimmune-deficient mice (Kim, K. J., Nature 362:841-844 (1993)). Further,a dominant-negative mutant of the VEGF receptor has been shown toinhibit growth of glioblastomas in mice.

Vascular permeability factor (VPF) has also been found to be responsiblefor persistent microvascular hyperpermeability to plasma proteins evenafter the cessation of injury, which is a characteristic feature ofnormal wound healing. This suggests that VPF is an important factor inwound healing. Brown, L. F. et al., J. Exp. Med. 176:1375-1379 (1992).

The expression of VEGF is high in vascularized tissues, (e.g., lung,heart, placenta and solid tumors) and correlates with angiogenesis bothtemporally and spatially. VEGF has also been shown to induceangiogenesis in vivo. Since angiogenesis is essential for the repair ofnormal tissues, especially vascular tissues, VEGF has been proposed foruse in promoting vascular tissue repair (e.g., in atherosclerosis).

U.S. Pat. No. 5,073,492, issued Dec. 17, 1991 to Chen et al., disclosesa method for synergistically enhancing endothelial cell growth in anappropriate environment which comprises adding to the environment, VEGF,effectors and serum-derived factor. Also, vascular endothelial cellgrowth factor C sub-unit DNA has been prepared by polymerase chainreaction techniques. The DNA encodes a protein that may exist as eithera heterodimer or homodimer. The protein is a mammalian vascularendothelial cell mitogen and, as such, is useful for the promotion ofvascular development and repair, as disclosed in European PatentApplication No. 92302750.2, published Sep. 30, 1992.

The Retina. The differentiated retina is composed of seven cell types:sensory (rod and cone photoreceptors), glia (Muller cells), and twotypes of neurons, intemeurons, (horizontal, bipolar, and amacrine), andprojection neurons (ganglion cells). The development of the various celltypes in the retina does not occur synchronously with the majority ofthe cones, and ganglion and horizontal cells developing before birth. Incontrast, differentiation of a majority of the rods, the main cell typein the rat retina, occurs postnatally. Clonal analysis of the progeny ofretinal precursor cells has demonstrated that these progenitor cells canproduce various combinations of retinal cell types indicating that atleast some of the progenitors are multipotential. Furthermore, findingsfrom both in vivo and in vitro studies suggest that the final phenotypeof the cell is largely lineage independent which suggest that thechanging microenvironment within the retina has a role in determiningthe cellular potential of the progenitor cells as well as thedifferentiated phenotype of the progeny.

In vitro, retinal cell proliferation and differentiation is regulated bya variety of factors, for example, FGF-2, CNTF, LIF, TGF, retinoic acid,and BDNF, as well as by extracellular matrix and cell adhesionmolecules, for example s-laminin. Yang and Cepko (J. Neurosci.16(19):6089-6099 (1996)) and more recently Wen et al. (J. Biol. Chem.273(4):2090-2097(1998)) have identified and characterized the expressionpattern of VEGFR-2/FLK-1, a member of the VEGF receptor family. VEGFRtranscripts are first detected at E11.5 in association with thedeveloping retinal vasculature and with the central region of the neuralretina (Yang and Cepko, J. Neurosci. 16(19):6089-6099 (1996)). Althoughit is not known if the two events are related, this developmental periodis also marked by the onset of ganglion cell development. Bydevelopmental day E15, VEGFR-2 expression extends to the periphery ofthe retina consistent with the outward gradient of retinal development.VEGFR-2 expression was largely localized to the ventricular zone duringthe perinatal period when neurogenesis is at its peak and a large numberof post-mitotic neurons are being formed.

The PDGF/VEGF superfamily currently includes 7 members. The 5 members ofthe VEGF sub-family bind to 4 different VEGF tyrosine kinase receptorswith distinct but overlapping specificities. VEGF, a 34-36 kDahomodimeric glycoprotein that is the prototypic family member, binds toVEGFR-1 and VEGFR-2. VEGF-B and VEGF-D bind only to VEGFR-1 or VEGFR-3,respectively. While VEGF-C, VEGF-2, has the highest affinity forVEGFR-3, it also binds with a lower affinity to VEGFR-2. Once activatedthe VEGF receptors tyrosine phosphorylate a number of proteinsdownstream in the signal transduction pathway includingphosphatidylinositol 3-kinase, phospholipase C, GAP, and Nck.

The hereditary retinal degenerative diseases (“HRD diseases”) are agroup of inherited conditions in which progressive, bilateraldegeneration of retinal structures leads to loss of retinal function;these diseases include, for example, age-related macular degeneration, aleading cause of visual impairment in the elderly; Leber's congenitalamaurosis, which causes its victims to be born blind; and retinitispigmentosa (“RP”). RP is the name given to those inherited retinopathieswhich are characterized by loss of retinal photoreceptors (rods andcones), with retinal electrical responses to light flashes (i.e.electroretinograms, or “ERGs”) that are reduced in amplitude. As thedisease progresses, patients show attenuated retinal arterioles, andfrequently show “bone spicule” pigmentation of the retina and waxypallor of the optic discs.

The incidence of RP in the United States is estimated to be about 1:3500births. Familial cases of RP usually present in childhood with nightblindness and loss of midperipheral visual field due to the loss of rodsin the peripheral retina. As the condition progresses, contraction ofthe visual fields eventually leads to blindness. Signs on fundusexamination in advanced stages include retinal vessel attenuation,intraretinal pigment in the peripheral fundus, and waxy pallor of theoptic disc. Patients have abnormal light-evoked electrical responsesfrom the retina (i.e., electroretinograms or ERGs), even in the earlystages in the absence of visible abnormalities on fundus examination.Histopathologic studies have revealed widespread loss of photoreceptorsin advanced stages. Therefore, there is a need in the art for methods oftreating photoreceptor cell disorders and injuries.

SUMMARY OF THE INVENTION

The polypeptides of the present invention have been identified as anovel vascular endothelial growth factor based on amino acid sequencehomology to human VEGF.

In accordance with one aspect of the present invention, there areprovided novel mature polypeptides, as well as biologically active anddiagnostically or therapeutically useful fragments, analogs, andderivatives thereof. The polypeptides of the present invention are ofhuman origin.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules comprising polynucleotidesencoding full length or truncated VEGF-2 polypeptides having the aminoacid sequences shown in SEQ ID NOS:2 or 4, respectively, or the aminoacid sequences encoded by the cDNA clones deposited in bacterial hostsas ATCC™ Deposit Number 97149 on May 12, 1995 or ATCC™ Deposit Number75698 on Mar. 4, 1994.

The present invention also relates to biologically active anddiagnostically or therapeutically useful fragments, analogs, andderivatives of VEGF-2.

In accordance with still another aspect of the present invention, thereare provided processes for producing such polypeptides by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a nucleic acid sequence encoding apolypeptide of the present invention, under conditions promotingexpression of said proteins and subsequent recovery of said proteins.

In accordance with yet another aspect of the present invention, thereare provided antibodies against such polypeptides and processes forproducing such antibodies.

In accordance with another aspect of the present invention, there areprovided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to nucleic acid sequences ofthe present invention.

In accordance with another aspect of the present invention, there areprovided methods of diagnosing diseases or a susceptibility to diseasesrelated to mutations in nucleic acid sequences of the present inventionand proteins encoded by such nucleic acid sequences.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

In accordance with yet a further aspect of the present invention, thereare provided processes for utilizing such polypeptides, orpolynucleotides encoding such polypeptides for therapeutic purposes, forexample, to protect or stimulate growth of photoreceptor cells.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be used, forexample, to inhibit or prevent photoreceptor growth.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E show the full length nucleotide (SEQ ID NO:1) and thededuced amino acid (SEQ ID NO:2) sequence of VEGF-2. The polypeptidecomprises approximately 419 amino acid residues of which approximately23 represent the leader sequence. The standard one letter abbreviationsfor amino acids are used. Sequencing was performed using the Model 373Automated DNA Sequencer (Applied Biosystems, Inc.). Sequencing accuracyis predicted to be greater than 97%.

FIGS. 2A-2D show the nucleotide (SEQ ID NO:3) and the deduced amino acid(SEQ ID NO:4) sequence of a truncated, biologically active form ofVEGF-2. The polypeptide comprises approximately 350 amino acid residuesof which approximately the first 24 amino acids represent the leadersequence.

FIGS. 3A-3B are an illustration of the amino acid sequence homologybetween PDGFa (SEQ ID NO:5), PDGFb (SEQ ID NO:6), VEGF (SEQ ID NO:7),and VEGF-2 (SEQ ID NO:4). The boxed areas indicate the conservedsequences and the location of the eight conserved cysteine residues.

FIG. 4 shows, in table-form, the percent homology between PDGFa, PDGFb,VEGF, and VEGF-2.

FIG. 5 shows the presence of VEGF-2 mRNA in human breast tumor celllines.

FIG. 6 depicts the results of a Northern blot analysis of VEGF-2 inhuman adult tissues.

FIG. 7 shows a photograph of an SDS-PAGE gel after in vitrotranscription, translation and electrophoresis of the polypeptide of thepresent invention. Lane 1: ¹⁴C and rainbow M. W. marker; Lane 2: FGFcontrol; Lane 3: VEGF-2 produced by M13-reverse and forward primers;Lane 4: VEGF-2 produced by M13 reverse and VEGF-F4 primers; Lane 5:VEGF-2 produced by M13 reverse and VEGF-F5 primers.

FIGS. 8A and 8B depict photographs of SDS-PAGE gels. VEGF-2 polypeptidewas expressed in a baculovirus system consisting of Sf9 cells. Proteinfrom the medium and cytoplasm of cells were analyzed by SDS-PAGE undernon-reducing (FIG. 8A) and reducing (FIG. 8B) conditions.

FIG. 9 depicts a photograph of an SDS-PAGE gel. The medium from Sf9cells infected with a nucleic acid sequence of the present invention wasprecipitated. The resuspended precipitate was analyzed by SDS-PAGE andstained with Coomassie brilliant blue.

FIG. 10 depicts a photograph of an SDS-PAGE gel. VEGF-2 was purifiedfrom the medium supernatant and analyzed by SDS-PAGE in the presence orabsence of the reducing agent b-mercaptoethanol and stained by Coomassiebrilliant blue.

FIG. 11 depicts reverse phase HPLC analysis of purified VEGF-2 using aRP-300 column (0.21×3 cm, Applied Biosystems, Inc.). The column wasequilibrated with 0.1% trifluoroacetic acid (Solvent A) and the proteinseluted with a 7.5 min gradient from 0 to 60% Solvent B, composed ofacetonitrile containing 0.07% TFA. The protein elution was monitored byabsorbance at 215 mn (“red” line) and 280 nm (“blue” line). Thepercentage of Solvent B is shown by the “green” line.

FIG. 12 shows a schematic representation of the pHE4-5 expression vector(SEQ ID NO:9) and the subcloned VEGF-2 cDNA coding sequence. Thelocations of the kanamycin resistance marker gene, the VEGF-2 codingsequence, the oriC sequence, and the lacIq coding sequence areindicated.

FIG. 13 shows the nucleotide sequence of the regulatory elements of thepHE promoter (SEQ ID NO:10). The two lac operator sequences, theShine-Delgamo sequence (S/D), and the terminal HindIII and NdeIrestriction sites (italicized) are indicated.

FIG. 14A-D shows that VEGF-2 treatment increases the level of rhodopsinprotein and the number of photoreceptor cells. Dissociated retinal cellswere prepared from P1 animals, plated at a density of 425 cells/mm² andtreated with VEGF-2 (A and B) or VEGF-2 (C and D). After 2 (opensquares), 5 (solid squares), 7 (open circles), or 9 (solid squares)days, the total number of cells in the cultures was estimated bymeasuring the calcein emission. The cultures were then fixed and thelevels of rhodopsin protein quantitated by ELISA.

FIG. 15 shows that the number of rhodopsin immunopositive cellsincreased as a function of VEGF-2 concentration. The retinal cells weremaintained in vitro for 8 days in the presence of either VEGF-1 orVEGF-2. The cultures were then fixed and immunohistochemically stainedfor rhodopsin.

FIG. 16A-C shows that VEGF-2 increases BrdU and [3H] thymidineincorporation in retinal cultures in a developmentally restrictedmanner. The cells were isolated from P1 animals and plated at a densityof 425 cells/mm². The cultures were initially treated 4 hours afterplating with either VEGF or VEGF-2. After 1, 2, or 3 days, the cultureswere labeled for 4 hours with BrdU. The cells were then fixed andprocessed for BrdU immunohistochemistry.

FIG. 17A-B shows the loss of the response to VEGF-2 or VEGF-1 as afunction of the time lapsed between the isolation of the cells and theinitial addition of the factors. One set of cultures was initiallytreated with factors 4 hours after plating (9/0) and subsequently,additional sets were treated after 24 or 48 hours (8/1 or 7/2,respectively). After 9 days in culture, the cells were fixed and thelevel of rhodopsin protein was quantitated by ELISA assay.

FIG. 18A-C shows VEGF increases the number of Amacrine but not Muller orEndothelial cells. Retinal cells were treated for 8 days with theindicated concentrations of VEGF-2. The cells were then fixed andimmunohistochemically stained for syntaxin (A), analyzed for the levelof high-affinity GABA uptake (B), or GFAP (C).

FIG. 19A-C shows the effect of developmental age on the response toVEGF-2. Retinal cells derived from E15 (A), E20 (B) or P1 (C) animalswere plated at a density of 212 (open squares), 318 (solid squares), or425 cells/mm². Four hours after plating, the cultures were treated withthe indicated concentrations of VEGF-2. After 24 hours, the cultureswere switched to serum-free medium and the factors were added again. Thecultures were then labeled with [3H] thymidine after 48 hours.

FIG. 20A-B compares the response of retinal cells to VEGF-2 and otherfactors. The cultures were seeded at a density of 425 cells/mm² andtreated for 9 days. Panel A shows the total number of cells in thecultures was estimated using calcein, while panel B shows the level ofrhodopsin protein determined by ELISA assay.

FIG. 21A-C shows that CNTF inhibits the response of the photoreceptorcell progenitors to VEGF-2. Retinal cultures were treated 24 hours afterplating with the indicated concentrations of CNTF in the presence orabsence of 150 ng/ml of VEGF-2. After 8 days in vitro, the amount ofrhodopsin protein was quantitated (A) and the total number of cells inthe cultures was determined (B). (C) To determine the effect of CNTFtreatment on the early proliferative response induced by VEGF-1, thecultures were treated with the indicated concentrations of VEGF-2 in thepresence or absence of 100 ng/ml CNTF. After 48 hours, the cultures werelabeled for 4 hours with [3H] thymidine.

FIG. 22 shows the enhanced LEC proliferation in response to VEGF-2 andantibody treatment.

FIG. 23 shows LEC proliferation in response to VEGF-2 andVEGF-2:antibody combination.

FIG. 24 shows the epitope map for murine anti VEGF-2 monoclonalantibodies.

FIG. 25 shows the status of the murine VEGF-2 monoclonal antibodies.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with one aspect of the present invention, there areprovided isolated nucleic acid molecules comprising a polynucleotideencoding a VEGF-2 polypeptide having the deduced amino acid sequence ofFIG. 1 (SEQ ID NO:2), which was determined by sequencing a cloned cDNA.The nucleotide sequence shown in SEQ ID NO:1 was obtained by sequencinga cDNA clone, which was deposited on May 12, 1995 at the American TypeTissue Collection (ATCC™), 10801 University Boulevard, Manassas, Va.20110-2209, and given ATCC™ Deposit No. 97149.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules comprising a polynucleotideencoding a truncated VEGF-2 polypeptide having the deduced amino acidsequence of FIG. 2 (SEQ ID NO:4), which was determined by sequencing acloned cDNA. The nucleotide sequence shown in SEQ ID NO:3 was obtainedby sequencing a cDNA clone, which was deposited on Mar. 4, 1994 at theAmerican Type Tissue Collection (ATCC™), 10801 University Boulevard,Manassas, Va. 20110-2209, and given ATCC™ Deposit Number 75698.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were predicted by translation of a DNA sequence determined asabove. Therefore, as is known in the art for any DNA sequence determinedby this automated approach, any nucleotide sequence determined hereinmay contain some errors. Nucleotide sequences determined by automationare typically at least about 90% identical, more typically at leastabout 95% to at least about 99.9% identical to the actual nucleotidesequence of the sequenced DNA molecule. The actual sequence can be moreprecisely determined by other approaches including manual DNA sequencingmethods well known in the art. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the predicted amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

A polynucleotide encoding a polypeptide of the present invention may beobtained from early stage human embryo (week 8 to 9) osteoclastomas,adult heart or several breast cancer cell lines. The polynucleotide ofthis invention was discovered in a cDNA library derived from early stagehuman embryo week 9. It is structurally related to the VEGF/PDGF family.It contains an open reading frame encoding a protein of about 419 aminoacid residues of which approximately the first 23 amino acid residuesare the putative leader sequence such that the mature protein comprises396 amino acids, and which protein exhibits the highest amino acidsequence homology to human vascular endothelial growth factor (30%identity), followed by PDGFa (24%) and PDGFb (22%). (See FIG. 4). It isparticularly important that all eight cysteines are conserved within allfour members of the family (see boxed areas of FIG. 3). In addition, thesignature for the PDGF/VEGF family, PXCVXXXRCXGCCN, (SEQ ID NO:8) isconserved in VEGF-2 (see FIG. 3). The homology between VEGF-2, VEGF andthe two PDGFs is at the protein sequence level. No nucleotide sequencehomology can be detected, and therefore, it would be difficult toisolate the VEGF-2 through simple approaches such as low stringencyhybridization.

The VEGF-2 polypeptide of the present invention is meant to include thefull length polypeptide and polynucleotide sequence which encodes forany leader sequences and for active fragments of the full lengthpolypeptide. Active fragments are meant to include any portions of thefull length amino acid sequence which have less than the full 419 aminoacids of the full length amino acid sequence as shown in SEQ ID NO:2,but still contain the eight cysteine residues shown conserved in FIG. 3and that still have VEGF-2 activity.

There are at least two alternatively spliced VEGF-2 MRNA sequencespresent in normal tissues. The two bands in FIG. 7, lane 5 indicate thepresence of the alternatively spliced mRNA encoding the VEGF-2polypeptide of the present invention.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1 or FIG. 2, or that of the depositedclones, or may be a different coding sequence which, as a result of theredundancy or degeneracy of the genetic code, encodes the same, maturepolypeptide as the DNA of FIG. 1, FIG. 2, or the deposited cDNAs.

The polynucleotide which encodes for the mature polypeptide of FIG. 1 orFIG. 2 or for the mature polypeptides encoded by the deposited cDNAs mayinclude: only the coding sequence for the mature polypeptide; the codingsequence for the mature polypeptide and additional coding sequences suchas a leader or secretory sequence or a proprotein sequence; the codingsequence for the mature polypeptide (and optionally additional codingsequences) and non-coding sequences, such as introns or non-codingsequence 5′ and/or 3′ of the coding sequence for the mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequences for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequences.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs, andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 or 2, or the polypeptide encoded by the cDNA of the depositedclones. The variant of the polynucleotide may be a naturally occurringallelic variant of the polynucleotide or a non-naturally occurringvariant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1 or 2 or the same maturepolypeptide encoded by the cDNA of the deposited clones as well asvariants of such polynucleotides which variants encode for a fragment,derivative, or analog of the polypeptides of FIG. 1 or 2, or thepolypeptide encoded by the cDNA of the deposited clones. Such nucleotidevariants include deletion variants, substitution variants, and additionor insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 or 2, or of the coding sequence of the deposited clones.As known in the art, an allelic variant is an alternate form of apolynucleotide sequence which have a substitution, deletion or additionof one or more nucleotides, which does not substantially alter thefunction of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide which aids in expression and secretion of apolypeptide from a host cell, for example, a leader sequence whichfunctions as a secretory sequence for controlling transport of apolypeptide from the cell. The polypeptide having a leader sequence is apreprotein and may have the leader sequence cleaved by the host cell toform the mature form of the polypeptide. The polynucleotides may alsoencode for a proprotein which is the mature protein plus additional 5′amino acid residues. A mature protein having a prosequence is aproprotein and is an inactive form of the protein. Once the prosequenceis cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and presequence (leadersequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell 37:767 (1984)).

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 95% identical, and more preferably at least 96%, 97%, 98% or 99%identical to (a) a nucleotide sequence encoding the polypeptide havingthe amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequenceencoding the polypeptide having the amino acid sequence in SEQ ID NO:2,but lacking the N-terminal methionine; (c) a nucleotide sequenceencoding the polypeptide having the amino acid sequence at positionsfrom about 1 to about 396 in SEQ ID NO:2; (d) a nucleotide sequenceencoding the polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC™ Deposit No. 97149; (e) a nucleotidesequence encoding the mature VEGF-2 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC™ Deposit No. 97149;or (f) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), or (e).

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 95% identical, and more preferably at least 96%, 97%, 98% or 99%identical to (a) a nucleotide sequence encoding the polypeptide havingthe amino acid sequence in SEQ ID NO:4; (b) a nucleotide sequenceencoding the polypeptide having the amino acid sequence in SEQ ID NO:4,but lacking the N-terminal methionine; (c) a nucleotide sequenceencoding the polypeptide having the amino acid sequence at positionsfrom about 1 to about 326 in SEQ ID NO:4; (d) a nucleotide sequenceencoding the polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC™ Deposit No. 75698; (e) a nucleotidesequence encoding the mature VEGF-2 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC™ Deposit No. 75698;or (f) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), or (e).

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a VEGF-2polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the VEGF-2polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5N or 3N terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in SEQ ID NOS:1 or 3, or to the nucleotidessequence of the deposited cDNA clone(s) can be determined conventionallyusing known computer programs such as the Bestfit program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2: 482-489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

The VEGF-2 variants may contain alterations in the coding regions,non-coding regions, or both. Especially preferred are polynucleotidevariants containing alterations which produce silent substitutions,additions, or deletions, but do not alter the properties or activitiesof the encoded polypeptide. Nucleotide variants produced by silentsubstitutions due to the degeneracy of the genetic code are preferred.Moreover, variants in which 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination are also preferred.VEGF-2 polynucleotide variants can be produced for a variety of reasons,e.g., to optimize codon expression for a particular host (change codonsin the human mRNA to those preferred by a bacterial host such as E.coli).

For example, site directed changes at the amino acid level of VEGF-2 canbe made by replacing a particular amino acid with a conservative aminoacid. Preferred conservative mutations include: M1 replaced with A, G,I, L, S, T, or V; H2 replaced with K, or R; S3 replaced with A, G, I, L,T, M, or V; L4 replaced with A, G, I, S, T, M, or V; G5 replaced with A,I, L, S, T, M, or V; F6 replaced with W, or Y; F7 replaced with W, or Y;S8 replaced with A, G, I, L, T, M, or V; V9 replaced with A, G, I, L, S,T, or M; A10 replaced with G, I, L, S, T, M, or V; S12 replaced with A,G, I, L, T, M, or V; L13 replaced with A, G, I, S, T, M, or V; L14replaced with A, G, I, S, T, M, or V; A15 replaced with G, I, L, S, T,M, or V; A16 replaced with G, I, L, S, T, M, or V; A17 replaced with G,I, L, S, T, M, or V; L18 replaced with A, G, I, S, T, M, or V; L19replaced with A, G, I, S, T, M, or V; G21 replaced with A, I, L, S, T,M, or V; R23 replaced with H, or K; E24 replaced with D; A25 replacedwith G, I, L, S, T, M, or V; A27 replaced with G, I, L, S, T, M, or V;A28 replaced with G, I, L, S, T, M, or V; A29 replaced with G, I, L, S,T, M, or V; A30 replaced with G, I, L, S, T, M, or V; A31 replaced withG, I, L, S, T, M, or V; F32 replaced with W, or Y; E33 replaced with D;S34 replaced with A, G, I, L, T, M, or V; G35 replaced with A, I, L, S,T, M, or V; L36 replaced with A, G, I, S, T, M, or V; D37 replaced withE; L38 replaced with A, G, I, S, T, M, or V; S39 replaced with A, G, I,L, T, M, or V; D40 replaced with E; A41 replaced with G, I, L, S, T, M,or V; E42 replaced with D; D44 replaced with E; A45 replaced with G, I,L, S, T, M, or V; G46 replaced with A,I, L, S, T, M, or V; E47 replacedwith D; A48 replaced with G, I, L, S, T, M, or V; T49 replaced with A,G, I, L, S, M, or V; A50 replaced with G, I, L, S, T, M, or V; Y51replaced with F, or W; A52 replaced with G, I, L, S, T, M, or V; S53replaced with A, G, I, L, T, M, or V; K54 replaced with H, or R; D55replaced with E; L56 replaced with A, G, I, S, T, M, or V; E57 replacedwith D; E58 replaced with D; Q59 replaced with N; L60 replaced with A,G, I, S, T, M, or V; R61 replaced with H, or K; S62 replaced with A, G,I, L, T, M, or V; V63 replaced with A, G, I, L, S, T, or M; S64 replacedwith A, G, I, L, T, M, or V; S65 replaced with A, G, I, L, T, M, or V;V66 replaced with A, G, I, L, S, T, or M; D67 replaced with E; E68replaced with D; L69 replaced with A, G, I, S, T, M, or V; M70 replacedwith A, G, I, L, S, T, or V; T71 replaced with A, G, I, L, S, M, or V;V72 replaced with A, G, I, L, S, T, or M; L73 replaced with A, G, I, S,T, M, or V; Y74 replaced with F, or W; E76 replaced with D; Y77 replacedwith F, or W; W78 replaced with F, or Y; K79 replaced with H, or R; M80replaced with A, G, I, L, S, T, or V; Y81 replaced with F, or W; K82replaced with H, or R; Q84 replaced with N; L85 replaced with A, G, I,S, T, M, or V; R86 replaced with H, or K; K87 replaced with H, or R; G88replaced with A, I, L, S, T, M, or V; G89 replaced with A, I, L, S, T,M, or V; W90 replaced with F, or Y; Q91 replaced with N; H92 replacedwith K, or R; N93 replaced with Q; R94 replaced with H, or K; E95replaced with D;Q96 replaced with N; A97 replaced with G, I, L, S, T, M,or V; N98 replaced with Q; L99 replaced with A, G, I, S, T, M, or V;N100 replaced with Q; S101 replaced with A, G, I, L, T, M, or V; R102replaced with H, or K; T103 replaced with A, G, I, L, S, M, or V; E104replaced with D; E105 replaced with D;T106 replaced with A, G, I, L, S,M, or V; I107 replaced with A, G, L, S, T, M, or V; K108 replaced withH, or R; F109 replaced with W, or Y; A110 replaced with G, I, L, S, T,M, or V; A111 replaced with G, I, L, S, T, M, or V; A112 replaced withG, I, L, S, T, M, or V; H113 replaced with K, or R;Y114 replaced with F,or W; N115 replaced with Q; T116 replaced with A, G, I, L, S, M, or V;E117 replaced with D; I118 replaced with A, G, L, S, T, M, or V; L119replaced with A, G, I, S, T, M, or V; K120 replaced with H, or R; S121replaced with A, G, I, L, T, M, or V; I122 replaced with A, G, L, S, T,M, or V; D123 replaced with E; N124 replaced with Q; E125 replaced withD; W126 replaced with F, or Y; R127 replaced with H, or K; K128 replacedwith H, or R; T129 replaced with A, G, I, L, S, M, or V; Q130 replacedwith N; M132 replaced with A, G, I, L, S, T, or V; R134 replaced with H,or K; E135replaced with D; V136 replaced with A, G, I, L, S, T, or M;I138 replaced with A, G, L, S, T, M, or V; D139 replaced with E; V140replaced with A, G, I, L, S, T, or M; G141 replaced with A, I, L, S, T,M, or V; K142 replaced with H, or R; E143 replaced with D; F144 replacedwith W, or Y; G145 replaced with A, I, L, S, T, M, or V; V146 replacedwith A, G, I, L, S, T, or M; A147 replaced with G, I, L, S, T, M, or V;T148 replaced with A, G, I, L, S, M, or V; N149 replaced with Q; T150replaced with A, G, I, L, S, M, or V; F151 replaced with W, or Y; F152replaced with W, or Y; K153 replaced with H, or R;V157 replaced with A,G, I, L, S, T, or M; S158 replaced with A, G, I, L, T, M, or V; V159replaced with A, G, I, L, S, T, or M; Y160 replaced with F, or W; R161replaced with H, or K; G163 replaced with A, I, L, S, T, M, or V; G164replaced with A, I, L, S, T, M, or V; N167 replaced with Q; S168replaced with A, G, I, L, T, M, or V; E169 replaced with D; G170replaced with A, I, L, S, T, M, or V; L171 replaced with A, G, I, S, T,M, or V; Q172 replaced with N; M174 replaced with A, G, I, L, S, T, orV; N175 replaced with Q; T176 replaced with A, G, I, L, S, M, or V; S177replaced with A, G, I, L, T, M, or V; T178 replaced with A, G, I, L, S,M, or V; S179 replaced with A, G, I, L, T, M, or V; Y180 replaced withF, or W; L181 replaced with A, G, I, S, T, M, or V; S182 replaced withA, G, I, L, T, M, or V; K183 replaced with H, or R; T184 replaced withA, G, I, L, S, M, or V; L185 replaced with A, G, I, S, T, M, or V; F186replaced with W, or Y; E187 replaced with D; I188 replaced with A, G, L,S, T, M, or V; T189 replaced with A, G, I, L, S, M, or V; V190 replacedwith A, G, I, L, S, T, or M; L192 replaced with A, G, I, S, T, M, or V;S193 replaced with A, G, I, L, T, M, or V; Q194 replaced with N; G195replaced with A, I, L, S, T, M, or V; K197 replaced with H, or R; V199replaced with A, G, I, L, S, T, or M; T200 replaced with A, G, I, L, S,M, or V; I201 replaced with A, G, L, S, T, M, or V; S202 replaced withA, G, I, L, T, M, or V; F203 replaced with W, or Y; A204 replaced withG, I, L, S, T, M, or V; N205 replaced with Q; H206 replaced with K, orR; T207 replaced with A, G, I, L, S, M, or V; S208 replaced with A, G,I, L, T, M, or V; R210 replaced with H, or K; M212 replaced with A, G,I, L, S, T, or V; S213 replaced with A, G, I, L, T, M, or V; K214replaced with H, or R; L215 replaced with A, G, I, S, T, M, or V; D216replaced with E; V217 replaced with A, G, I, L, S, T, or M; Y218replaced with F, or W; R219 replaced with H, or K;Q220 replaced with N;V221 replaced with A, G, I, L, S, T, or M; H222 replaced with K, or R;S223 replaced with A, G, I, L, T, M, or V; I224 replaced with A, G, L,S, T, M, or V; I225 replaced with A, G, L, S, T, M, or V; R226 replacedwith H, or K; R227 replaced with H, or K; S228 replaced with A, G, I, L,T, M, or V; L229 replaced with A, G, I, S, T, M, or V; A231 replacedwith G, I, L, S, T, M, or V; T232 replaced with A, G, I, L, S, M, or V;L233 replaced with A, G, I, S, T, M, or V; Q235 replaced with N; Q237replaced with N; A238 replaced with G, I, L, S, T, M, or V; A239replaced with G, I, L, S, T, M, or V; N240 replaced with Q; K241replaced with H, or R; T242 replaced with A, G, I, L, S, M, or V; T245replaced with A, G, I, L, S, M, or V;N246 replaced with Q; Y247 replacedwith F, or W; M248 replaced with A, G, I, L, S, T, or V; W249 replacedwith F, or Y; N250 replaced with Q; N251 replaced with Q; H252 replacedwith K, or R; 1253 replaced with A, G, L, S, T, M, or V; R255 replacedwith H, or K; L257 replaced with A, G, I, S, T, M, or V; A258 replacedwith G, I, L, S, T, M, or V; Q259 replaced with N; E260 replaced with D;D261 replaced with E; F262 replaced with W, or Y; M263 replaced with A,G, I, L, S, T, or V; F264 replaced with W, or Y; S265 replaced with A,G, I, L, T, M, or V; S266 replaced with A, G, I, L, T, M, or V;D267replaced with E; A268 replaced with G, I, L, S, T, M, or V; G269replaced with A, I, L, S, T, M, or V; D270 replaced with E; D271replaced with E;S272 replaced with A, G, I, L, T, M, or V; T273 replacedwith A, G, I, L, S, M, or V; D274 replaced with E; G275 replaced with A,I, L, S, T, M, or V;F276 replaced with W, or Y; H277 replaced with K, orR; D278 replaced with E; I279 replaced with A, G, L, S, T, M, or V; G281replaced with A, I, L, S, T, M, or V; N283 replaced with Q; K284replaced with H, or R; E285 replaced with D; L286 replaced with A, G, I,S, T, M, or V; D287 replaced with E; E288 replaced with D; E289 replacedwith D; T290 replaced with A, G, I, L, S, M, or V; Q292 replaced with N;V294 replaced with A, G, I, L, S, T, or M; R296 replaced with H, or K;A297 replaced with G, I, L, S, T, M, or V; G298 replaced with A, I, L,S, T, M, or V; L299 replaced with A, G, I, S, T, M, or V; R300 replacedwith H, or K; A302 replaced with G, I, L, S, T, M, or V; S303 replacedwith A, G, I, L, T, M, or V; G305 replaced with A, I, L, S, T, M, or V;H307 replaced with K, or R; K308 replaced with H, or R; E309 replacedwith D; L310 replaced with A, G, I, S, T, M, or V; D311 replaced withE;R312 replaced with H, or K; N313 replaced with Q; S314 replaced withA, G, I, L, T, M, or V; Q316 replaced with N; V318 replaced with A, G,I, L, S, T, or M; K320 replaced with H, or R; N321 replaced with Q; K322replaced with H, or R; L323 replaced with A, G, I, S, T, M, or V; F324replaced with W, or Y; S326 replaced with A, G, I, L, T, M, or V; Q327replaced with N; G329 replaced with A, I, L, S, T, M, or V; A330replaced with G, I, L, S, T, M, or V; N331 replaced with Q; R332replaced with H, or K; E333 replaced with D; F334 replaced with W, or Y;D335 replaced with E; E336 replaced with D; N337 replaced with Q; T338replaced with A, G, I, L, S, M, or V; Q340 replaced with N; V342replaced with A, G, I, L, S, T, or M; K344 replaced with H, or R; R345replaced with H, or K; T346 replaced with A, G, I, L, S, M, or V; R349replaced with H, or K; N350 replaced with Q; Q351 replaced with N;L353replaced with A, G, I, S, T, M, or V; N354 replaced with Q; G356replaced with A, I, L, S, T, M, or V; K357 replaced with H, or R; A359replaced with G, I, L, S, T, M, or V; E361 replaced with D; T363replaced with A, G, I, L, S, M, or V; E364 replaced with D; S365replaced with A, G, I, L, T, M, or V; Q367 replaced with N; K368replaced with H, or R; L370 replaced with A, G, I, S, T, M, or V; L371replaced with A, G, I, S, T, M, or V; K372 replaced with H, or R; G373replaced with A, I, L, S, T, M, or V; K374 replaced with H, or R; K375replaced with H, or R; F376 replaced with W, or Y; H377replaced with K,or R; H378 replaced with K, or R; Q379 replaced with N; T380 replacedwith A, G, I, L, S, M, or V; S382 replaced with A, G, I, L, T, M, or V;Y384 replaced with F, or W; R385 replaced with H, or K; R386 replacedwith H, or K; T389 replaced with A, G, I, L, S, M, or V; N390 replacedwith Q; R391 replaced with H, or K; Q392 replaced with N; K393 replacedwith H, or R; A394 replaced with G, I, L, S, T, M, or V; E396 replacedwith D; G398 replaced with A, I, L, S, T, M, or V; F399 replaced with W,or Y; S400 replaced with A, G, I, L, T, M, or V; Y401 replaced with F,or W; S402 replaced with A, G, I, L, T, M, or V; E403 replaced with D;E404 replaced with D; V405 replaced with A, G, I, L, S, T, or M; R407replaced with H, or K; V409 replaced with A, G, I, L, S, T, or M; S411replaced with A, G, I, L, T, M, or V; Y412 replaced with F, or W; W413replaced with F, or Y; Q414 replaced with N; R415 replaced with H, or K;Q417 replaced with N; M418 replaced with A, G, I, L, S, T, or V; and/orS419 replaced with A, G, I, L, T, M, or V of FIGS. 1A-1E.

The resulting constructs can be routinely screened for activities orfunctions described throughout the specification and known in the art.Preferably, the resulting constructs have an increased and/or adecreased VEGF-2 activity or function, while the remaining VEGF-2activities or functions are maintained. More preferably, the resultingconstructs have more than one increased and/or decreased VEGF-2 activityor function, while the remaining VEGF-2 activities or functions aremaintained.

Besides conservative amino acid substitution, variants of VEGF-2 include(i) substitutions with one or more of the non-conserved amino acidresidues, where the substituted amino acid residues may or may not beone encoded by the genetic code, or (ii) substitution with one or moreof amino acid residues having a substituent group, or (iii) fusion ofthe mature polypeptide with another compound, such as a compound toincrease the stability and/or solubility of the polypeptide (forexample, polyethylene glycol), or (iv) fusion of the polypeptide withadditional amino acids, such as, for example, an IgG Fc fusion regionpeptide, or leader or secretory sequence, or a sequence facilitatingpurification. Such variant polypeptides are deemed to be within thescope of those skilled in the art from the teachings herein.

For example, VEGF-2 polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

For example, preferred non-conservative substitutions of VEGF-2 include:M1 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H2 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S3 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L4 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; G5 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; F6 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; F7 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; S8 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V9replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A10 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; C11 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; S12 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; L13 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L14 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;A15 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A16 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A17 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; L18 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L19 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;P20 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or C; G21 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P22replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; R23 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; E24 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; A25 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P26replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; A27 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A28 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A29 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; A30 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; A31 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;F32 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;E33 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; S34 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G35 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L36 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; D37 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; L38 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; S39 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; D40 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; A41 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E42replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;P43 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or C; D44 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; A45 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G46replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E47 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A48 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; T49 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; A50 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; Y51 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,M, V, P, or C; A52 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S53 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K54 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D55 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L56replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E57 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E58 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q59replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;L60 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R61 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S62 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V63 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; S64 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; S65 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V66 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D67 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E68replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;L69 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M70 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; T71 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; V72 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L73 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;Y74 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;P75 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or C; E76 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; Y77 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; W78 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; K79 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; M80 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y81replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;K82replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;C83 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or P; Q84 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,P, or C; L85 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R86replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K87replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G88replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G89 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; W90 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; Q91 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; H92 replaced with D, E, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N93 replaced with D, E, H, K,R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R94 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E95 replaced with H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q96 replaced with D, E,H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A97 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; N98 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, F, W, Y, P, or C; L99 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; N100 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, F, W, Y, P, or C; S101 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; R102 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; T103 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;E104 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; E105 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; T106 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I107replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K108 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F109 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A110 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A111 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; A112 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; H113 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; Y114 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; N 115 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; T116 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;E117 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; I118 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L119replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K120 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S121 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; I122 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; D123 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; N124 replaced with D, E, H, K, R, A, G, I, L,S, T, M, V, F, W, Y, P, or C; E125 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; W126 replaced with D, E, H, K, R, N, Q,A, G, I, L, S, T, M, V, P, or C; R127 replaced with D, E, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; K128 replaced with D, E, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; T129 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; Q130 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, F, W, Y, P, or C; C131 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, or P; M132 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; P133 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, or C; R134 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; E135 replaced with H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; V136 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; C137 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, or P; I138 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;D139 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; V140 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G141replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K142 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E143 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F144 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G145 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V146 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; A147 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; T148 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;N149 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; T150 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F151replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F152replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K153replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P154replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; P155 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or C; C156 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, or P; V157 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S158 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V159 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; Y160 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R161 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C162 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; G163 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; G164 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; C165 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, or P; C166 replaced with D, E, H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, or P; N167 replaced with D, E, H, K, R, A, G,I, L, S, T, M, V, F, W, Y, P, or C; S168 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; E169 replaced with H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; G170 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; L171 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q172replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;C173 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or P; M174 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N175replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;T176 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S177 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; T178 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; S179 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; Y180 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,M, V, P, or C; L181 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S182 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K183 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T184 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L185 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; F186 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; E187 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; I188 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; T189 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; V190 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P191replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; L192 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S193replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q194 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G195 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; P196 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K197 replaced with D,E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P198 replaced with D,E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; V199 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; T200 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; I201 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; S202 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;F203 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;A204 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N205 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; H206replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T207replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S208 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; C209 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; R210 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C211 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; M212 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; S213 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; K214 replaced with D, E, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; L215 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; D216 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; V217 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y218replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R219replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q220replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;V221 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H222 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S223 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; I224 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; I225 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; R226 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; R227 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; S228 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L229 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P230 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A231replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T232 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L233 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; P234 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or C; Q235 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C; C236 replaced with D, E, H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, or P; Q237 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; A238 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; A239 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; N240 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; K241 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; T242 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;C243 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or P; P244 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, or C; T245 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;N246 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; Y247 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, orC; M248 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W249replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; N250replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;N251 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; H252 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; I253 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C254replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; R255 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; C256 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or P; L257 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A258replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q259 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E260 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D261replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;F262 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;M263 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F264 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S265 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; S266 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; D267 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; A268 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; G269 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; D270 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; D271 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; S272 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T273replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D274 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G275 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; F276 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; H277 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D278 replaced with H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I279 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; C280 replaced with D, E, H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, or P;G281 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; P282 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or C; N283 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C; K284 replaced with D, E, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; E285 replaced with H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; L286 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; D287 replaced with H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; E288 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; E289 replaced with H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; T290 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; C291 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or P; Q292 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, F, W, Y, P, or C; C293 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or P; V294 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; C295 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or P; R296 replaced with D, E, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; A297 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; G298 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L299replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R300 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P301 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A302replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S303 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; C304 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; G305 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; P306 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; H307 replaced with D, E, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; K308 replaced with D, E, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; E309 replaced with H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; L310 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; D311 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; R312 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; N313 replaced with D, E, H, K, R, A, G, I, L,S, T, M, V, F, W, Y, P, or C; S314 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; C315 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or P; Q316 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, F, W, Y, P, or C; C317 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or P; V318 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; C319 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or P; K320 replaced with D, E, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; N321 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, F, W, Y, P, or C; K322 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; L323 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; F324 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; P325 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, or C; S326 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;Q327 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; C328 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or P; G329 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A330replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N331 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R332 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E333 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F334replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D335replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;E336 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; N337 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; T338 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C339replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; Q340 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; C341 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, or P; V342 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;C343 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or P; K344 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; R345 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; T346 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C347replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; P348 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or C; R349 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; N350 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,Y, P, or C; Q351 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; P352 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or C; L353 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; N354 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,P, or C; P355 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, or C; G356 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;K357 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;C358 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or P; A359 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C360replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; E361 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; C362 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, or P; T363 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;E364 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; S365 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P366replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; Q367 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; K368 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; C369 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, or P; L370 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L371 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K372 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G373 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; K374 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K375 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F376 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; H377 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H378 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q379 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T380 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; C381 replaced with D, E, H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, or P; S382 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; C383 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or P; Y384 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; R385 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; R386 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; P387 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; C388 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, or P; T389 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; N390 replaced with D, E, H, K, R, A, G, I, L,S, T, M, V, F, W, Y, P, or C; R391 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; Q392 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C; K393 replaced with D, E, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; A394 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; C395 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, or P; E396 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; P397 replaced with D, E, H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, or C; G398 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; F399 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,T, M, V, P, or C; S400 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; Y401 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, orC; S402 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E403replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;E404 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; V405 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C406replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; R407 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; C408 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or P; V409 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P410replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; S411 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y412replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; W413replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q414replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;R415 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;P416 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or C; Q417 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,P, or C; M418 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;and/or S419 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C of FIGS.1A-1E.

The resulting constructs can be routinely screened for activities orfunctions described throughout the specification and known in the art.Preferably, the resulting constructs have an increased and/or decreasedVEGF-2 activity or function, while the remaining VEGF-2 activities orfunctions are maintained. More preferably, the resulting constructs havemore than one increased and/or decreased VEGF-2 activity or function,while the remaining VEGF-2 activities or functions are maintained.

As described in detail below, the polypeptides of the present inventioncan be used to raise polyclonal and monoclonal antibodies, which areuseful in diagnostic assays for detecting VEGF-2 protein expression oras agonists and antagonists capable of enhancing or inhibiting VEGF-2protein function. Further, such polypeptides can be used in the yeasttwo-hybrid system to “capture” VEGF-2 protein binding proteins which arealso candidate agonist and antagonist according to the presentinvention. The yeast two hybrid system is described in Fields and Song,Nature 340:245-246 (1989).

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. These immunogenic epitopes arebelieved to be confined to a few loci on the molecule. On the otherhand, a region of a protein molecule to which an antibody can bind isdefined as an “antigenic epitope.” The number of immunogenic epitopes ofa protein generally is less than the number of antigenic epitopes. See,for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002(1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G. et al., (1983)Science 219:660-666. Peptides capable of eliciting protein-reactive seraare frequently represented in the primary sequence of a protein, can becharacterized by a set of simple chemical rules, and are confinedneither to immunodominant regions of intact proteins (i.e., immunogenicepitopes) nor to the amino or carboxyl terminals. Peptides that areextremely hydrophobic and those of six or fewer residues generally areineffective at inducing antibodies that bind to the mimicked protein;longer, soluble peptides, especially those containing proline residues,usually are effective. Sutcliffe et al., supra, at 661. For instance, 18of 20 peptides designed according to these guidelines, containing 8-39residues covering 75% of the sequence of the influenza virushemagglutinin HA1 polypeptide chain, induced antibodies that reactedwith the HA1 protein or intact virus; and 12/12 peptides from the MuLVpolymerase and 18/18 from the rabies glycoprotein induced antibodiesthat precipitated the respective proteins.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. Thus, a highproportion of hybridomas obtained by fusion of spleen cells from donorsimmunized with an antigen epitope-bearing peptide generally secreteantibody reactive with the native protein. Sutcliffe et al., supra, at663. The antibodies raised by antigenic epitope-bearing peptides orpolypeptides are useful to detect the mimicked protein, and antibodiesto different peptides may be used for tracking the fate of variousregions of a protein precursor which undergoes post-translationalprocessing. The peptides and anti-peptide antibodies may be used in avariety of qualitative or quantitative assays for the mimicked protein,for instance in competition assays since it has been shown that evenshort peptides (e.g., about 9 amino acids) can bind and displace thelarger peptides in immunoprecipitation assays. See, for instance, Wilsonet al., Cell 37:767-778 (1984) at 777. The anti-peptide antibodies ofthe invention also are useful for purification of the mimicked protein,for instance, by adsorption chromatography using methods well known inthe art.

Antigenic epitope-bearing peptides and polypeptides of the inventiondesigned according to the above guidelines preferably contain a sequenceof at least seven, more preferably at least nine and most preferablybetween about 15 to about 30 amino acids contained within the amino acidsequence of a polypeptide of the invention. However, peptides orpolypeptides comprising a larger portion of an amino acid sequence of apolypeptide of the invention, containing about 30, 40, 50, 60, 70, 80,90, 100, or 150 amino acids, or any length up to and including theentire amino acid sequence of a polypeptide of the invention, also areconsidered epitope-bearing peptides or polypeptides of the invention andalso are useful for inducing antibodies that react with the mimickedprotein. Preferably, the amino acid sequence of the epitope-bearingpeptide is selected to provide substantial solubility in aqueoussolvents (i.e., the sequence includes relatively hydrophilic residuesand highly hydrophobic sequences are preferably avoided); and sequencescontaining proline residues are particularly preferred.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate VEGF-2-specific antibodies include the following: apolypeptide comprising amino acid residues from about leu-37 to aboutGlu-45 in SEQ ID NO:2, from about Tyr-58 to about Gly-66 in SEQ ID NO:2,from about Gln-73 to about Glu-81 in SEQ ID NO:2, from about Asp-100 toabout Cys-108 in SEQ ID NO:2, from about Gly-140 to about Leu-148 in SEQID NO:2, from about Pro-168 to about Val-176 in SEQ ID NO:2, from aboutHis-183 to about Lys-191 in SEQ ID NO:2, from about Ile-201 to aboutThr-209 in SEQ ID NO:2, from about Ala-216 to about Tyr-224 in SEQ IDNO:2, from about Asp-244 to about His-254 in SEQ ID NO:2, from aboutGly-258 to about Glu-266 in SEQ ID NO:2, from about Cys-272 to aboutSer-280 in SEQ ID NO:2, from about Pro-283 to about Ser-291 in SEQ IDNO:2, from about Cys-296 to about Gln-304 in SEQ ID NO:2, from aboutAla-307 to about Cys-316 in SEQ ID NO:2, from about Val-319 to aboutCys-335 in SEQ ID NO:2, from about Cys-339 to about Leu-347 in SEQ IDNO:2, from about Cys-360 to about Glu-373 in SEQ ID NO:2, from aboutTyr-378 to about Val-386 in SEQ ID NO:2, and from about Ser-388 to aboutSer-396 in SEQ ID NO:2. These polypeptide fragments have been determinedto bear antigenic epitopes of the VEGF-2 protein by the analysis of theJameson-Wolf antigenic index.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means for making peptides or polypeptidesincluding recombinant means using nucleic acid molecules of theinvention. For instance, a short epitope-bearing amino acid sequence maybe fused to a larger polypeptide which acts as a carrier duringrecombinant production and purification, as well as during immunizationto produce anti-peptide antibodies. Epitope-bearing peptides also may besynthesized using known methods of chemical synthesis. For instance,Houghten has described a simple method for synthesis of large numbers ofpeptides, such as 10-20 mg of 248 different 13 residue peptidesrepresenting single amino acid variants of a segment of the HA1polypeptide which were prepared and characterized (by ELISA-type bindingstudies) in less than four weeks. Houghten, R. A. (1985) G Proc. Natl.Acad. Sci. USA 82:5131-5135. This “Simultaneous Multiple PeptideSynthesis (SMPS)” process is further described in U.S. Pat. No.4,631,211 to Houghten et al. (1986). In this procedure the individualresins for the solid-phase synthesis of various peptides are containedin separate solvent-permeable packets, enabling the optimal use of themany identical repetitive steps involved in solid-phase methods. Acompletely manual procedure allows 500-1000 or more syntheses to beconducted simultaneously. Houghten et al., supra, at 5134.

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art. See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. etal., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J.Gen. Virol. 66:2347-2354 (1985). Generally, animals may be immunizedwith free peptide; however, anti-peptide antibody titer may be boostedby coupling of the peptide to a macromolecular carrier, such as keyholelimpet hemacyanin (KLH) or tetanus toxoid. For instance, peptidescontaining cysteine may be coupled to carrier using a linker such asm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carrier using a more general linking agentsuch as glutaraldehyde.

Animals such as rabbits, rats and mice are immunized with either free orcarrier-coupled peptides, for instance, by intraperitoneal and/orintradermal injection of emulsions containing about 100 mg peptide orcarrier protein and Freund's adjuvant. Several booster injections may beneeded, for instance, at intervals of about two weeks, to provide auseful titer of anti-peptide antibody which can be detected, forexample, by ELISA assay using free peptide adsorbed to a solid surface.The titer of anti-peptide antibodies in serum from an immunized animalmay be increased by selection of anti-peptide antibodies, for instance,by adsorption to the peptide on a solid support and elution of theselected antibodies according to methods well known in the art.

Immunogenic epitope-bearing peptides of the invention, i.e., those partsof a protein that elicit an antibody response when the whole protein isthe immunogen, are identified according to methods known in the art. Forinstance, Geysen et al., supra, discloses a procedure for rapidconcurrent synthesis on solid supports of hundreds of peptides ofsufficient purity to react in an enzyme-linked immunosorbent assay.Interaction of synthesized peptides with antibodies is then easilydetected without removing them from the support. In this manner apeptide bearing an immunogenic epitope of a desired protein may beidentified routinely by one of ordinary skill in the art. For instance,the immunologically important epitope in the coat protein offoot-and-mouth disease virus was located by Geysen et al. with aresolution of seven amino acids by synthesis of an overlapping set ofall 208 possible hexapeptides covering the entire 213 amino acidsequence of the protein. Then, a complete replacement set of peptides inwhich all 20 amino acids were substituted in turn at every positionwithin the epitope were synthesized, and the particular amino acidsconferring specificity for the reaction with antibody were determined.Thus, peptide analogs of the epitope-bearing peptides of the inventioncan be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen(1987) further describes this method of identifying a peptide bearing animmunogenic epitope of a desired protein.

Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describes ageneral method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a Amimotope) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C₁-C₇-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

As one of skill in the art will appreciate, VEGF-2 polypeptides of thepresent invention and the epitope-bearing fragments thereof describedabove can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86(1988)). In accordance with the present invention, novel variants ofVEGF-2 are also described. These can be produced by deleting orsubstituting one or more amino acids of VEGF-2. Natural mutations arecalled allelic variations. Allelic variations can be silent (no changein the encoded polypeptide) or may have altered amino acid sequence.

In order to attempt to improve or alter the characteristics of nativeVEGF-2, protein engineering may be employed. Recombinant DNA technologyknown to those skilled in the art can be used to create novelpolypeptides. Muteins and deletions can show, e.g., enhanced activity orincreased stability. In addition, they could be purified in higher yieldand show better solubility at least under certain purification andstorage conditions. Set forth below are examples of mutations that canbe constructed.

Amino Terminal and Carboxy Terminal Deletions

Furthermore, VEGF-2 appears to be proteolytically cleaved uponexpression resulting in polypeptide fragments of the following sizeswhen run on a SDS-PAGE gel (sizes are approximate) (See, FIGS. 6-8, forexample): 80, 59, 45, 43, 41, 40, 39, 38, 37, 36, 31, 29, 21, and 15kDa. These polypeptide fragments are the result of proteolytic cleavageat both the N-terminal and C-terminal portions of the protein. Theseproteolytically generated fragments appears to have activity,particularly the 21 kDa fragment.

In addition, protein engineering may be employed in order to improve oralter one or more characteristics of native VEGF-2. The deletion ofcarboxy terminal amino acids can enhance the activity of proteins. Oneexample is interferon gamma that shows up to ten times higher activityby deleting ten amino acid residues from the carboxy terminus of theprotein (Döbeli et al., J. of Biotechnology 7:199-216 (1988)). Thus, oneaspect of the invention is to provide polypeptide analogs of VEGF-2 andnucleotide sequences encoding such analogs that exhibit enhancedstability (e.g., when exposed to typical pH, thermal conditions or otherstorage conditions) relative to the native VEGF-2 polypeptide.Particularly, preferred VEGF-2 polypeptides are shown below (numberingstarts with the first amino acid in the protein (Met) (FIG. 1 (SEQ IDNO:2)):

Ala (residue 24 25) to Ser (residue 419); Pro (25 26) to Ser (419); Ala(26 27) to Ser (419); Ala (27 28) to Ser (419); Ala (28 29) to Ser(419); Ala (29 30) to Ser (419); Ala (30 31) to Ser (419); Phe (31 32)to Ser (419); Glu (32 33) to Ser (419); Ser (33 34) to Ser (419); Gly(34 35) to Ser (419); Leu (35 36) to Ser (419); Asp (36 37) to Ser(419); Leu (37 38) to (Ser (419); Ser (38 39) to Ser (419); Asp (39 40)to Ser (419); Ala (40 41) to Ser (419); Glu (41 42) to Ser (419); Pro(42 43) to Ser (419); Asp (43 44) to Ser (419); Ala (44 45) to Ser(419); Gly (45 46) to Ser (419); Glu (46 47) to Ser (419); Ala (47 48)to Ser (419); Thr (48 49) to Ser (419); Ala (49 50) to Ser (419); Tyr(50 51) to Ser (419); Ser (52 53) to Ser (419); Asp (54 55) to Ser(419); Val (62 63) to Ser (419); Val (65 66) to Ser (419); Met(1), Glu(23 24), or Ala (24 25) to Met (418); Met (1), Glu (23 24), or Ala (2425) to Gln (417); Met (1), Glu (23 24), or Ala (24 25) to Pro (416);Met(1), Glu (23 24), or Ala (24 25) to Arg (415); Met(1), Glu (23 24),or Ala (24 25) to Gln (414); Met(1), Glu (23 24), or Ala (24 25) to Trp(413); Met(1), Glu (23 24), or Ala (24 25) to Tyr (412); Met(1), Glu (2324), or Ala (24 25) to Ser (411); Met(1), Glu (23 24), or Ala (24 25) toPro (410); Met(1), Glu (23 24), or Ala (24 25) to Val (409); Met(1), Glu(23 24), or Ala (24 25) to Cys (408); Met(1), Glu (23 24), or Ala (2425) to Arg (407); Met(1), Glu (23 24), or Ala (24 25) to Cys (406);Met(1), Glu (23 24), or Ala (24 25) to Val (405); Met(1), Glu (23 24),or Ala (24 25) to Glu (404); Met(1), Glu (23 24), or Ala (24 25) to Glu(403); Met(1), Glu (23 24), or Ala (24 25) to Ser (402); Met(1), Glu (2324), or Ala (24 25) to Gly (398); Met(1), Glu (23 24), or Ala (24 25) toPro (397); Met(1), Glu (23 24), or Ala (24 25) to Lys (393); Met(1), Glu(23 24), or Ala (24 25) to Met(263); Met(1), Glu (23 24), or Ala (24 25)to Asp(311); Met(1), Glu (23 24), or Ala (24 25) to Pro (367 366);Met(1) to Ser (419); Met(1) to Ser(228); Glu(47) to Ser(419); Ala(111)to Lys(214); Ala(112) to Lys(214); His(113) to Lys(214); Tyr(114) toLys(214); Asn(115) to Lys(214); Thr(116) to Lys(214); Thr(103) toLeu(215); Glu(104) to Leu(215); Glu(105) to Leu(215); Thr(106) toLeu(215); Ile(107) to Leu(215); Lys(108) to Leu(215); Phe(109) toLeu(215); Ala(110) to Leu(215); Ala(111) to Leu(215); Ala(112) toLeu(215); His(113) to Leu(215); Tyr(114) to Leu(215); Asn(115) toLeu(215); Thr(116) to Leu(215); Thr(103) to Ser(228); Glu(104) toSer(228); Glu(105) to Ser(228); Thr(106) to Ser(228); Ile(107) toSer(228); Lys(108) to Ser(228); Phe(109) to Ser(228); Ala(110) toSer(228); Ala(111) to Ser(228); Ala(112) to Ser(228); His(113) toSer(228); Tyr(114) to Ser(228); Asn(115) to Ser(228); Thr(116) toSer(228); Thr(103) to Leu(229); Glu(104) to Leu(229); Thr(103) toArg(227); Glu(104) to Arg(227); Glu(105) to Arg (227); Thr(106) to Arg(227); Ile(107) to Arg (227); Lys(108) to Arg (227); Phe(109) to Arg(227); Ala(110) to Arg (227); Ala(111) to Arg (227); Ala(112) to Arg(227); His(113) to Arg (227); Tyr(114) to Arg (227); Asn(115) to Arg(227); Thr(116) to Arg (227); Thr(103) to Ser(213); Glu(104) toSer(213); Glu(105) to Ser(213); Thr(106) to Ser(213); Ile(107) toSer(213); Lys(108) to Ser(213); Phe(109) to Ser(213); Ala(110) toSer(213); Ala(111) to Ser(213); Ala(112) to Ser(213); His(113) to);Tyr(114) to Ser(213); Asn(115) to Ser(213); Thr(116) to Ser(213);Thr(103) to Lys(214); Glu(104) to Lys(214); Glu(105) to Lys(214);Thr(106) to Lys(214); Ile(107) to Lys(214); Lys(108) to Lys(214);Phe(109) to Lys(214); Ala(110) to Lys(214); Glu(105) to Leu(229);Thr(106) to Leu(229); Ile(107) to Leu(229); Lys(108) to Leu(229);Phe(109) to Leu(229); Ala(110) to Leu(229); Ala(111) to Leu(229);Ala(112) to Leu(229); His(113) to Leu(229); Tyr(114) to Leu(229);Asn(115) to Leu(229); Thr(116) to Leu(229).

Preferred embodiments include the following deletion mutants:Thr(103)-Arg(227); Glu(104)-Arg(227); Ala(112)-Arg (227);Thr(103)-Ser(213); Glu(104)-Ser(213); Thr(103)-Leu(215);Glu(47)-Ser(419); Met(1), Glu (23 24), or Ala (24 25)-Met(263); Met(1),Glu (23 24), or Ala (24 25)-Asp(311); Met(1), Glu (23 24), or Ala (2425)-Pro (367 366); Met(1)-Ser(419); and Met(1)-Ser(228) of (FIG. 1 (SEQID NO:2)).

Also included by the present invention are deletion mutants having aminoacids deleted from both the NB terminus and the C-terminus. Such mutantsinclude all combinations of the N-terminal deletion mutants andC-terminal deletion mutants described above. Those combinations can bemade using recombinant techniques known to those skilled in the art.

Particularly, N-terminal deletions of the VEGF-2 polypeptide can bedescribed by the general formula m-396, where m is an integer from −23to 388, where m corresponds to the position of the amino acid residueidentified in SEQ ID NO:2. Preferably, N-terminal deletions retain theconserved boxed area of FIG. 3 (PXCVXXXRCXGCCN)(SEQ ID NO: 8), andinclude polypeptides comprising the amino acid sequence of residues: A-2to S-396; P-3 to S-396; A-4 to S-396; A-5 to S-396; A-6 to S-396; A-7 toS-396; A-8 to S-396; F-9 to S-396; E-10 to S-396; S-11 to S-396; G-12 toS-396; L-13 to S-396; D-14 to S-396; L-15 to S-396; S-16 to S-396; D-17to S-396; A-18 to S-396; E-19 to S-396; P-20 to S-396; D-21 to S-396;A-22 to S-396; G-23 to S-396; E-24 to S-396; A-25 to S-396; T-26 toS-396; A-27 to S-396; Y-28 to S-396; A-29 to S-396; S-30 to S-396; K-31to S-396; D-32 to S-396; L-33 to S-396; E-34 to S-396; E-35 to S-396;Q-36 to S-396; L-37 to S-396; R-38 to S-396; S-39 to S-396; V-40 toS-396; S-41 to S-396; S-42 to S-396; V-43 to S-396; D-44 to S-396; E-45to S-396; L-46 to S-396; M-47 to S-396; T-48 to S-396; V-49 to S-396;L-50 to S-396; Y-51 to S-396; P-52 to S-396; E-53 to S-396; Y-54 toS-396; W-55 to S-396; K-56 to S-396; M-57 to S-396; Y-58 to S-396; K-59to S-396; C-60 to S-396; Q-61 to S-396; L-62 to S-396; R-63 to S-396;K-64 to S-396; G-65 to S-396; G-66 to S-396; W-67 to S-396; Q-68 toS-396; H-69 to S-396; N-70 to S-396; R-71 to S-396; E-72 to S-396; Q-73to S-396; A-74 to S-396; N-75 to S-396; L-76 to S-396; N-77 to S-396;S-78 to S-396; R-79 to S-396; T-80 to S-396; E-81 to S-396; E-82 toS-396; T-83 to S-396; I-84 to S-396; K-85 to S-396; F-86 to S-396; A-87to S-396; A-88 to S-396; A-89 to S-396; H-90 to S-396; Y-91 to S-396;N-92 to S-396; T-93 to S-396; E-94 to S-396; I-95 to S-396; L-96 toS-396; K-97 to S-396; S-98 to S-396; I-99 to S-396; D-100 to S-396;N-101 to S-396; E-102 to S-396; W-103 to S-396; R-104 to S-396; K-105 toS-396; T-106 to S-396; Q-107 to S-396; C-108 to S-396; M-109 to S-396;P-110 to S-396; R-111 to S-396; E-112 to S-396; V-113 to S-396; C-114 toS-396; I-115 to S-396; D-116 to S-396; V-117 to S-396; G-118 to S-396;K-119 to S-396; E-120 to S-396; F-121 to S-396; G-122 to S-396; V-123 toS-396; A-124 to S-396; T-125 to S-396; N-126 to S-396; T-127 to S-396;F-128 to S-396; F-129 to S-396; K-130 to S-396; P-131 to S-396 of SEQ IDNO:2. Also preferred are polynucleotides encoding these N-terminaldeletion mutants.

Moreover, C-terminal deletions of the VEGF-2 polypeptide can also bedescribed by the general formula −23-n, where n is an integer from −15to 395 where n corresponds to the position of amino acid residueidentified in SEQ ID NO:2. Preferably, C-terminal deletions retain theconserved boxed area of FIG. 3 (PXCVXXXRCXGCCN)(SEQ ID NO: 8), andinclude polypeptides comprising the amino acid sequence of residues: E-1to M-395; E-1 to Q-394; E-1 to P-393; E-1 to R-392; E-1 to Q-391; E-1 toW-390; E-1 to Y-389; E-1 to S-388; E-1 to P-387; E-1 to V-386; E-1 toC-385; E-1 to R-384; E-1 to C-383; E-1 to V-382; E-1 to E-381; E-1 toE-380; E-1 to S-379; E-1 to Y-378; E-1 to S-377; E-1 to F-376; E-1 toG-375; E-1 to P-374; E-1 to E-373; E-1 to C-372; E-1 to A-371; E-1 toK-370; E-1 to Q-369; E-1 to R-368; E-1 to N-367; E-1 to T-366; E-1 toC-365; E-1 to P-364; E-1 to R-363; E-1 to R-362; E-1 to Y-361; E-1 toC-360; E-1 to S-359; E-1 to C-358; E-1 to T-357; E-1 to Q-356; E-1 toH-355; E-1 to H-354; E-1 to F-353; E-1 to K-352; E-1 to K-351; E-1 toG-350; E-1 to K-349; E-1 to L-348; E-1 to L-347; E-1 to C-346; E-1 toK-345; E-1 to Q-344; E-1 to P-343; E-1 to S-342; E-1 to E-341; E-1 toT-340; E-1 to C-339; E-1 to E-338; E-1 to C-337; E-1 to A-336; E-1 toC-335; E-1 to K-334; E-1 to G-333; E-1 to P-332; E-1 to N-331; E-1 toL-330; E-1 to P-329; E-1 to Q-328; E-1 to N-327; E-1 to R-326; E-1 toP-325; E-1 to C-324; E-1 to T-323; E-1 to R-322; E-1 to K-321; E-1 toC-320; E-1 to V-319; E-1 to C-318; E-1 to Q-317; E-1 to C-316; E-1 toT-315; E-1 to N-314; E-1 to E-313; E-1 to D-312; E-1 to F-311; E-1 toE-310; E-1 to R-309; E-1 to N-308; E-1 to A-307; E-1 to G-306; E-1 toC-305; E-1 to Q-304; E-1 to S-303; E-1 to P-302; E-1 to F-301; E-1 toL-300; E-1 to K-299; E-1 to N-298; E-1 to K-297; E-1 to C-296; E-1 toV-295; E-1 to C-294; E-1 to Q-293; E-1 to C-292; E-1 to S-291; E-1 toN-290; E-1 to R-289; E-1 to D-288; E-1 to L-287; E-1 to E-286; E-1 toK-285; E-1 to H-284; E-1 to P-283; E-1 to G-282; E-1 to C-281; E-1 toS-280; E-1 to A-279; E-1 to P-278; E-1 to R-277; E-1 to L-276; E-1 toG-275; E-1 to A-274; E-1 to R-273; E-1 to C-272; E-1 to V-271; E-1 toC-270; E-1 to Q-269; E-1 to C-268; E-1 to T-267; E-1 to E-266; E-1 toE-265; E-1 to D-264; E-1 to L-263; E-1 to E-262; E-1 to K-261; E-1 toN-260; E-1 to P-259; E-1 to G-258; E-1 to C-257; E-1 to I-256; E-1 toD-255; E-1 to H-254; E-1 to F-253; E-1 to G-252; E-1 to D-251; E-1 toT-250; E-1 to S-249; E-1 to D-248; E-1 to D-247; E-1 to G-246; E-1 toA-245; E-1 to D-244; E-1 to S-243; E-1 to S-242; E-1 to F-241; E-1 toM-240; E-1 to F-239; E-1 to D-238; E-1 to E-237; E-1 to Q-236; E-1 toA-235; E-1 to L-234; E-1 to C-233; E-1 to R-232; E-1 to C-231; E-1 to1-230; E-1 to H-229; E-1 to N-228; E-1 to N-227; E-1 to W-226; E-1 toM-225; E-1 to Y-224; E-1 to N-223; E-1 to T-222; E-1 to P-221; E-1 toC-220; E-1 to T-219; E-1 to K-218; E-1 to N-217; E-1 to A-216; E-1 toQ-214; E-1 to C-213; E-1 to Q-212; E-1 to P-211; E-1 to L-210; E-1 toT-209; E-1 to A-208; E-1 to P-207; E-1 to L-206; E-1 to S-205; E-1 toR-204; E-1 to R-203; E-1 to I-202; E-1 to I-201; E-1 to S-200; E-1 toH-199; E-1 to V-198; E-1 to Q-197; E-1 to R-196; E-1 to Y-195; E-1 toV-194; E-1 to D-193; E-1 to L-192; E-1 to K-191; E-1 to S-190; E-1 toM-189; E-1 to C-188; E-1 to R-187; E-1 to C-186; E-1 to S-185; E-1 toT-184; E-1 to H-183; E-1 to N-182; E-1 to A-181; E-1 to F-180; E-1 toS-179; E-1 to I-178; E-1 to T-177; E-1 to V-176; E-1 to P-175; E-1 toK-174; E-1 to P-173; E-1 to G-172; E-1 to Q-171; E-1 to S-170; E-1 toL-169; E-1 to P-168; E-1 to V-167; E-1 to T-166; E-1 to I-165; E-1 toE-164; E-1 to F-163; E-1 to L-162; E-1 to T-161; E-1 to K-160; E-1 toS-159; E-1 to L-158; E-1 to Y-157; E-1 to S-156; E-1 to T-155; E-1 toS-154; E-1 to T-153; E-1 to N-152; E-1 to M-151; E-1 to C-150; E-1 toQ-149; E-1 to L-148; E-1 to G-147; E-1 to E-146; E-1 to S-145; E-1 toN-144; of SEQ ID NO:2. Also preferred are polynucleotides encoding theseC-terminal deletion mutants. Preferably, any of the above listed N- orC-terminal deletions can be combined to produce a N- and C-terminaldeleted VEGF-2 polypeptide, which retains the conserved box domain.

Moreover, the invention also provides polypeptides having one or moreamino acids deleted from both the amino and the carboxyl termini, whichmay be described generally as having residues m-n of SEQ ID NO:2, wheren and m are integers as described above.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:1 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1660 of SEQID NO:1, b is an integer of 15 to 1674, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:1, and where theb is greater than or equal to a+14.

Thus, in one aspect, N-terminal deletion mutants are provided by thepresent invention. Such mutants include those comprising the amino acidsequence shown in FIG. 1 (SEQ ID NO:2) except for a deletion of at leastthe first 24 N-terminal amino acid residues (i.e., a deletion of atleast Met (1)-Glu (24)) but not more than the first 115 N-terminal aminoacid residues of FIG. 1 (SEQ ID NO:2). Alternatively, first 24N-terminal amino acid residues (i.e., a deletion of at least Met (1)-Glu(24)) but not more than the first 103 N-terminal amino acid residues ofFIG. 1 (SEQ ID NO:2), etc., etc.

In another aspect, C-terminal deletion mutants are provided by thepresent invention. Such mutants include those comprising the amino acidsequence shown in FIG. 1 (SEQ ID NO:2) except for a deletion of at leastthe last C-terminal amino acid residue (Ser (419)) but not more than thelast 220 C-terminal amino acid residues (i.e., a deletion of amino acidresidues Val (199)-Ser (419)) of FIG. 1 (SEQ ID NO:2). Alternatively,the deletion will include at least the last C-terminal amino acidresidue but not more than the last 216 C-terminal amino acid residues ofFIG. 1 (SEQ ID NO:2). Alternatively, the deletion will include at leastthe last C-terminal amino acid residue but not more than the last 204C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2). Alternatively,the deletion will include at least the last C-terminal amino acidresidues but not more than the last 192 C-terminal amino acid residuesof FIG. 1 (SEQ ID NO:2).

Alternatively, the deletion will include at least the last C-terminalamino acid residues but not more than the last 156 C-terminal amino acidresidues of FIG. 1 (SEQ ID NO:2). Alternatively, the deletion willinclude at least the last C-terminal amino acid residues but not morethan the last 108 C-terminal amino acid residues of FIG. 1 (SEQ IDNO:2). Alternatively, the deletion will include at least the lastC-terminal amino acid residues but not more than the last 52 C-terminalamino acid residues of FIG. 1 (SEQ ID NO:2).

In yet another aspect, also included by the present invention aredeletion mutants having amino acids deleted from both the N-terminal andC-terminal residues. Such mutants include all combinations of theN-terminal deletion mutants and C-terminal deletion mutants describedabove.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatednucleic acid molecule having the nucleotide sequence of the depositedcDNA(s) or the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3is intended fragments at least about 15 nt, and more preferably at leastabout 20 nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments of 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675,700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025,1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325,1350, 1375, 1400, 1425, 1450, 1475, 1500, 1525, 1550, 1575, 1600, 1625,1650 or 1674 nt in length are also useful according to the presentinvention as are fragments corresponding to most, if not all, of thenucleotide sequence of the deposited cDNA(s) or as shown in SEQ ID NO:1or SEQ ID NO:3. By a fragment at least 20 nt in length, for example, isintended fragments which include 20 or more contiguous bases from thenucleotide sequence of the deposited cDNA(s) or the nucleotide sequenceas shown in SEQ ID NOS:1 or 3.

Moreover, representative examples of VEGF-2 polynucleotide fragmentsinclude, for example, fragments having a sequence from about nucleotidenumber 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350,351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800,800-850, 851-900, 901-950, or 951 to the end of SEQ ID NO:1 or the cDNAcontained in the deposited clone. In this context “about” includes theparticularly recited ranges, larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini. Preferably,these fragments encode a polypeptide which has biological activity.

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, 50 or more bases.The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promoter regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or MRNA to determine whichmembers of the library the probe hybridizes to.

A VEGF-2 “polynucleotide” also includes those polynucleotides capable ofhybridizing, under stringent hybridization conditions, to sequencescontained in SEQ ID NO:1 or for instance, the cDNA clone(s) contained inATCC™ Deposit Nos. 97149 or 75698, the complement thereof. “Stringenthybridization conditions” refers to an overnight incubation at 42° C. ina solution comprising 50% formamide, 5× SSC (750 mM NaCl, 75 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

Also contemplated are nucleic acid molecules that hybridize to theVEGF-2 polynucleotides at lower stringency hybridization conditions.Changes in the stringency of hybridization and signal detection areprimarily accomplished through the manipulation of formamideconcentration (lower percentages of formamide result in loweredstringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37° C. in asolution comprising 6×SSPE (20×SSPE =3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA;followed by washes at 50° C. with 1×SSPE, 0.1% SDS. In addition, toachieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA shown in the sequencelisting), or to a complementary stretch of T (or U) residues, would notbe included in the definition of “polynucleotide,” since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 nt of the reference polynucleotide. These are useful asdiagnostic probes and primers as discussed above and in more detailbelow.

By a portion of a polynucleotide of “at least 20 nt in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in SEQ ID NO:1). Of course, apolynucleotide which hybridizes only to a poly A sequence (such as the3N terminal poly(A) tract of the VEGF-2 cDNA shown in SEQ ID NOS:1 or3), or to a complementary stretch of T (or U) resides, would not beincluded in a polynucleotide of the invention used to hybridize to aportion of a nucleic acid of the invention, since such a polynucleotidewould hybridize to any nucleic acid molecule containing a poly (A)stretch or the complement thereof (e.g., practically any double-strandedcDNA clone).

The present application is directed to nucleic acid molecules at least95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shownin SEQ ID NOS:1 or 3 or to the nucleic acid sequence of the depositedcDNA(s), irrespective of whether they encode a polypeptide having VEGF-2activity. This is because even where a particular nucleic acid moleculedoes not encode a polypeptide having VEGF-2 activity, one of skill inthe art would still know how to use the nucleic acid molecule, forinstance, as a hybridization probe or a polymerase chain reaction (PCR)primer. Uses of the nucleic acid molecules of the present invention thatdo not encode a polypeptide having VEGF-2 activity include, inter alia,(1) isolating the VEGF-2 gene or allelic variants thereof in a cDNAlibrary; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide precise chromosomal location of theVEGF-2 gene, as described in Verma et al., Human Chromosomes: A Manualof Basic Techniques, Pergamon Press, New York (1988); and Northern Blotanalysis for detecting VEGF-2 MRNA expression in specific tissues.Preferred, however, are nucleic acid molecules having sequences at least95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence shown inSEQ ID NOS:1 or 3 or to a nucleic acid sequence of the deposited cDNA(s)which do, in fact, encode a polypeptide having VEGF-2 protein activity.By “a polypeptide having VEGF-2 activity” is intended polypeptidesexhibiting VEGF-2 activity in a particular biological assay. Forexample, VEGF-2 protein activity can be measured using, for example,mitogenic assays and endothelial cell migration assays. See, e.g.,Olofsson et al., Proc. Natl. Acad. Sci. USA 93:2576-2581 (1996) andJoukov et al., EMBO J. 5:290-298 (1996).

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence of the depositedcDNA(s) or the nucleic acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3will encode a polypeptide “having VEGF-2 protein activity.” In fact,since degenerate variants of these nucleotide sequences all encode thesame polypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving VEGF-2 protein activity. This is because the skilled artisan isfully aware of amino acid substitutions that are either less likely ornot likely to significantly effect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), wherein the authors indicate that proteinsare surprisingly tolerant of amino acid substitutions.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95%, 96%, 97%, or 98% identity to a polynucleotide which encodesthe polypeptides of SEQ ID NOS:2 or 4, as well as fragments thereof,which fragments have at least 30 bases and preferably at least 50 basesand to polypeptides encoded by such polynucleotides.

“Identity” per se has an art-recognized meaning and can be calculatedusing published techniques. (See, e.g.: (Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, (1988);Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, (1993); Computer Analysis of Sequence Data,Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, NewJersey, (1994); Sequence Analysis in Molecular Biology, von Heinje, G.,Academic Press, (1987); and Sequence Analysis Primer, Gribskov, M. andDevereux, J., eds., M Stockton Press, New York, (1991).) While thereexists a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans. (Carillo, H., and Lipton, D., SIAM J. AppliedMath. 48:1073 (1988).) Methods commonly employed to determine identityor similarity between two sequences include, but are not limited to,those disclosed in “Guide to Huge Computers,” Martin J. Bishop, ed.,Academic Press, San Diego, (1994), and Carillo, H., and Lipton, D., SIAMJ. Applied Math. 48:1073 (1988). Methods for aligning polynucleotides orpolypeptides are codified in computer programs, including the GCGprogram package (Devereux, J., et al., Nucleic Acids Research 12(1):387(1984)), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol.215:403 (1990), Bestfit program (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, University Research Park,575 Science Drive, Madison, Wis. 53711 (using the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics2:482-489 (1981)). By a polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence encoding theVEGF-2 polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may bean entire sequence SEQ ID NO:1, the ORF (open reading frame), or anyfragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to anucleotide sequence of the presence invention can be determinedconventionally using known computer programs. A preferred method fordetermining the best overall match between a query sequence (a sequenceof the present invention) and a subject sequence, also referred to as aglobal sequence alignment, can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al. (Comp. App. Biosci.6:237-245 (1990)). In a sequence alignment the query and subjectsequences are both DNA sequences. An RNA sequence can be compared byconverting U's to T's. The result of said global sequence alignment isin percent identity. Preferred parameters used in a FASTDB alignment ofDNA sequences to calculate percent identity are: Matrix=Unitary,k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization GroupLength=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, WindowSize=500 or the length of the subject nucleotide sequence, whichever isshorter. If the subject sequence is shorter than the query sequencebecause of 5′ or 3′ deletions, not because of internal deletions, amanual correction must be made to the results. This is because theFASTDB program does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.Whether a nucleotide is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thiscorrected score is what is used for the purposes of the presentinvention. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, (indels) or substituted withanother amino acid. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequences shown in SEQ ID NOs:2 or 4 or to the amino acid sequenceencoded by deposited DNA clone can be determined conventionally usingknown computer programs. A preferred method for determining the bestoverall match between a query sequence (a sequence of the presentinvention) and a subject sequence, also referred to as a global sequencealignment, can be determined using the FASTDB computer program based onthe algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245).In a sequence alignment the query and subject sequences are either bothnucleotide sequences or both amino acid sequences. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2,Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0,Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

VEGF-2 Polypeptides

The present invention further relates to polypeptides which have thededuced amino acid sequence of FIG. 1 or 2, or which has the amino acidsequence encoded by the deposited cDNAs, as well as fragments, analogs,and derivatives of such polypeptides.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptide of FIG. 1 or 2 or that encoded by the deposited cDNA, meansa polypeptide which retains the conserved motif of VEGF proteins asshown in FIG. 3 and essentially the same biological function oractivity.

In the present invention, a “polypeptide fragment” refers to a shortamino acid sequence contained in SEQ ID NO:2 or encoded by the cDNAcontained in the deposited clone. Protein fragments may be“free-standing,” or comprised within a larger polypeptide of which thefragment forms a part or region, most preferably as a single continuousregion. Representative examples of polypeptide fragments of theinvention, include, for example, fragments from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-180,181-200, 201-220, 221-240, 241-260, 261-280, or 281 to the end of thecoding region. Moreover, polypeptide fragments can be about 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids inlength. In this context “about” includes the particularly recitedranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, ateither extreme or at both extremes.

Preferred polypeptide fragments include the secreted VEGF-2 protein aswell as the mature form. Further preferred polypeptide fragments includethe secreted VEGF-2 protein or the mature form having a continuousseries of deleted residues from the amino or the carboxy terminus, orboth. For example, any number of amino acids, ranging from 1-60, can bedeleted from the amino terminus of either the secreted VEGF-2polypeptide or the mature form. Similarly, any number of amino acids,ranging from 1-30, can be deleted from the carboxy terminus of thesecreted VEGF-2 protein or mature form. Furthermore, any combination ofthe above amino and carboxy terminus deletions are preferred. Similarly,polynucleotide fragments encoding these VEGF-2 polypeptide fragments arealso preferred.

Also preferred are VEGF-2 polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Polypeptide fragments of SEQ ID NO:2 falling withinconserved domains are specifically contemplated by the presentinvention. (See FIG. 2.) Moreover, polynucleotide fragments encodingthese domains are also contemplated.

Other preferred fragments are biologically active VEGF-2 fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the VEGF-2 polypeptide. Thebiological activity of the fragments may include an improved desiredactivity, or a decreased undesirable activity.

The polypeptides of the present invention may be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides,preferably recombinant polypeptides.

It will be recognized in the art that some amino acid sequences of theVEGF-2 polypeptide can be varied without significant effect of thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity.

Thus, the invention further includes variations of the VEGF-2polypeptide which show substantial VEGF-2 polypeptide activity or whichinclude regions of VEGF-2 protein such as the protein portions discussedbelow. Such mutants include deletions, insertions, inversions, repeats,and type substitutions. As indicated above, guidance concerning whichamino acid changes are likely to be phenotypically silent can be foundin Bowie, J. U., et al., “Deciphering the Message in Protein Sequences:Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990).

Thus, the fragments, derivatives, or analogs of the polypeptides of FIG.1 or 2, or that encoded by the deposited cDNAs may be: (I) one in whichone or more of the amino acid residues are substituted with a conservedor non-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code; or (ii) one in which one or more of theamino acid residues includes a substituent group; or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol); or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence; or (v) one in which comprisesfewer amino acid residues shown in SEQ ID NOS: 2 or 4, and retains theconserved motif and yet still retains activity characteristics of theVEGF family of polypeptides. Such fragments, derivatives, and analogsare deemed to be within the scope of those skilled in the art from theteachings herein.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the VEGF-2 protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361:266-268(1993) describes certain mutations resulting in selective binding ofTNF-a to only one of the two known types of TNF receptors. Thus, theVEGF-2 of the present invention may include one or more amino acidsubstitutions, deletions or additions, either from natural mutations orhuman manipulation.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Tables 1 and 2).

TABLE 1 Conservative Amino Acid Substitutions Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

TABLE 2 Preferred Amino Acid Substitutions Original Preferred ResidueSubstitutions Exemplary Substitutions Ala (A) Val Val; Leu; Ile Arg (R)Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Lys; Arg Asp (D) Glu Glu Cys (C)Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Arg Asn;Gln; Lys; Arg Ile (I) Leu Leu; Val; Met; Ala; Phe; norleucine Leu (L)Ile norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met(M) Leu Leu; Phe; Ile Phe (F) Leu Leu; Val; Ile; Ala Pro (P) Gly Gly Ser(S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Phe Trp; Phe; Thr;Ser Val (V) Leu Ile; Leu; Met; Phe; Ala; norleucine

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of substitutions for any given VEGF-2polypeptide will not be more than 50, 40, 30, 25, 20, 15, 10, 5 or 3.

Amino acids in the VEGF-2 protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro, or in vitro proliferativeactivity. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

In specific embodiments, the polynucleotides of the invention are lessthan 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, or 7.5 kb in length.In a further embodiment, polynucleotides of the invention comprise atleast 15 contiguous nucleotides of VEGF-2 coding sequence, but do notcomprise all or a portion of any VEGF-2 intron. In another embodiment,the nucleic acid comprising VEGF-2 coding sequence does not containcoding sequences of a genomic flanking gene (i.e., 5′ or 3′ to theVEGF-2 gene in the genome).

The polypeptides of the present invention include the polypeptides ofSEQ ID NOS:2 and 4 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least 70%identity) to the polypeptides of SEQ ID NOS:2 and 4, and more preferablyat least 90% similarity (more preferably at least 95% identity) to thepolypeptides of SEQ ID NOS:2 and 4, and still more preferably at least95% similarity (still more preferably at least 90% identity) to thepolypeptides of SEQ ID NOS:2 and 4 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids. Asknown in the art “similarity” between two polypeptides is determined bycomparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The polypeptides of the present invention include the polypeptideencoded by the deposited cDNA including the leader; the maturepolypeptide encoded by the deposited the cDNA minus the leader (i.e.,the mature protein); a polypeptide comprising amino acids about −23 toabout 396 in SEQ ID NO:2; a polypeptide comprising amino acids about −22to about 396 in SEQ ID NO:2; a polypeptide comprising amino acids about1 to about 396 in SEQ ID NO:2; as well as polypeptides which are atleast 95% identical, and more preferably at least 96%, 97%, 98% or 99%identical to the polypeptides described above and also include portionsof such polypeptides with at least 30 amino acids and more preferably atleast 50 amino acids.

VEGF-2 Derivatives

The VEGF-2 wild type and analogs may be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties may improve the solubility, the biological halflife or absorption of the protein. The moieties may also reduce oreliminate any desirable side effects of the proteins and the like. anoverview for those moieties can be found in REMINGTON'S PHARMACEUTICALSCIENCES, 18th ed., Mack Publishing Co., Easton, Pa. (1990).

The chemical moieties most suitable for derivatization include watersoluble polymers. A water soluble polymer is desirable because theprotein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. Preferably, thepolymer will be pharmaceutically acceptable for the preparation of atherapeutic product or composition. One skilled in the art will be ableto select the desired polymer based on such considerations as whetherthe polymer/protein conjugate will be used therapeutically, and if so,the desired dosage, circulation time, resistance to proteolysis, andother considerations. The effectiveness of the derivatization may beascertained by administering the derivative, in the desired form (i.e.,by osmotic pump, or, more preferably, by injection or infusion, or,further formulated for oral, pulmonary or other delivery routes), anddetermining its effectiveness.

Suitable water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weightranges from about 2 kDa to about 100 kDa for ease in handling andmanufacturing (the term “about” indicating that in preparations ofpolyethylene glycol, some molecules will weigh more, some less, than thestated molecular weight). Other sizes may be used, depending on thedesired therapeutic profile (e.g., the duration of sustained releasedesired, the effects, if any on biological activity, the ease inhandling, the degree or lack of antigenicity and other known effects ofpolyethylene glycol on a therapeutic protein or variant).

The number of polymer molecules so attached may vary, and one skilled inthe art will be able to ascertain the effect on function. One maymono-derivatize, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to protein (or peptide)molecules will vary, as will their concentrations in the reactionmixture. In general, the optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted protein or polymer) willbe determined by factors such as the desired degree of derivatization(e.g., mono, di-, tri-, etc.), the molecular weight of the polymerselected, whether the polymer is branched or unbranched, and thereaction conditions.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art. See for example, EP 0 401384, the disclosure of which is hereby incorporated by reference(coupling PEG to G-CSF), see also Malik et al., Exp. Hematol20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresylchloride). For example, polyethylene glycol may be covalently boundthrough amino acid residues via a reactive group, such as, a free aminoor carboxyl group. Reactive groups are those to which an activatedpolyethylene glycol molecule may be bound. The amino acid residueshaving a free amino group may include lysine residues and the N-terminalamino acid residue. Those having a free carboxyl group may includeaspartic acid residues, glutamic acid residues, and the C-terminal aminoacid residue. Sulfhydryl groups may also be used as a reactive group forattaching the polyethylene glycol molecule(s). For therapeutic purposes,attachment at an amino group, such as attachment at the N-terminus orlysine group is preferred. Attachment at residues important for receptorbinding should be avoided if receptor binding is desired.

One may specifically desire an N-terminal chemically modified protein.Using polyethylene glycol as an illustration of the presentcompositions, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective N-terminal chemicalmodification may be accomplished by reductive alkylation which exploitsdifferential reactivity of different types of primary amino groups(lysine versus the N-terminal) available for derivatization in aparticular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved. For example, onemay selectively N-terminally pegylate the protein by performing thereaction at a pH which allows one to take advantage of the pKadifferences between the epsilon-amino group of the lysine residues andthat of the alpha-amino group of the N-terminal residue of the protein.By such selective derivatization, attachment of a water soluble polymerto a protein is controlled: the conjugation with the polymer takes placepredominantly at the N-terminus of the protein and no significantmodification of other reactive groups, such as the lysine side chainamino groups, occurs. Using reductive alkylation, the water solublepolymer may be of the type described above, and should have a singlereactive aldehyde for coupling to the protein. Polyethylene glycolpropionaldehyde, containing a single reactive aldehyde, may be used.

The present invention contemplates use of derivatives which areprokaryote-expressed VEGF-2, or variants thereof, linked to at least onepolyethylene glycol molecule, as well as use of VEGF-2, or variantsthereof, attached to one or more polyethylene glycol molecules via anacyl or alkyl linkage.

Pegylation may be carried out by any of the pegylation reactions knownin the art. See, for example: Focus on Growth Factors, 3 (2): 4-10(1992); EP 0 154 316, the disclosure of which is hereby incorporated byreference; EP 0 401 384; and the other publications cited herein thatrelate to pegylation. The pegylation may be carried out via an acylationreaction or an alkylation reaction with a reactive polyethylene glycolmolecule (or an analogous reactive water-soluble polymer).

Pegylation by acylation generally involves reacting an active esterderivative of polyethylene glycol with the VEGF-2 protein or variant.Any known or subsequently discovered reactive PEG molecule may be usedto carry out the pegylation of VEGF-2 protein or variant. A preferredactivated PEG ester is PEG esterified to N-hydroxysuccinimide. As usedherein, “acylation” is contemplated to include without limitation thefollowing types of linkages between the therapeutic protein and a watersoluble polymer such as PEG: amide, carbamate, urethane, and the like.See Bioconjugate Chem. 5:133-140 (1994). Reaction conditions may beselected from any of those known in the pegylation art or thosesubsequently developed, but should avoid conditions of temperature,solvent, and pH that would inactivate the VEGF-2 or variant to bemodified.

Pegylation by acylation will generally result in a poly-pegylated VEGF-2protein or variant. Preferably, the connecting linkage will be an amide.Also preferably, the resulting product will be substantially only(e.g., >95%) mono-, di- or tri-pegylated. However, some species withhigher degrees of peglylation may be formed in amounts depending on thespecific reaction conditions used. If desired, more purified pegylatedspecies may be separated from the mixture, particularly unreactedspecies, by standard purification techniques, including, among others,dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gelfiltration chromatography and electrophoresis.

Pegylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with the VEGF-2 protein or variant in the presence ofa reducing agent. Pegylation by alkylation can also result inpoly-pegylated VEGF-2 protein or variant. In addition, one canmanipulate the reaction conditions to favor pegylation substantiallyonly at the a-amino group of the N-terminus of the VEGF-2 protein orvariant (i.e., a mono-pegylated protein). In either case ofmonopegylation or polypegylation, the PEG groups are preferably attachedto the protein via a —CH2-NH— group. With particular reference to the—CH2- group, this type of linkage is referred to herein as an “alkyl”linkage.

Derivatization via reductive alkylation to produce a monopegylatedproduct exploits differential reactivity of different types of primaryamino groups (lysine versus the N-terminal) available forderivatization. The reaction is performed at a pH which allows one totake advantage of the pKa differences between the ε-amino groups of thelysine residues and that of the α-amino group of the N-terminal residueof the protein. By such selective derivatization, attachment of a watersoluble polymer that contains a reactive group such as an aldehyde, to aprotein is controlled: the conjugation with the polymer takes placepredominantly at the N-terminus of the protein and no significantmodification of other reactive groups, such as the lysine side chainamino groups, occurs. In one important aspect, the present inventioncontemplates use of a substantially homogeneous preparation ofmonopolymer/VEGF-2 protein (or variant) conjugate molecules (meaningVEGF-2 protein or variant to which a polymer molecule has been attachedsubstantially only (i.e., >95%) in a single location). Morespecifically, if polyethylene glycol is used, the present invention alsoencompasses use of pegylated VEGF-2 protein or variant lacking possiblyantigenic linking groups, and having the polyethylene glycol moleculedirectly coupled to the VEGF-2 protein or variant.

Thus, it is contemplated that VEGF-2 to be used in accordance with thepresent invention may include pegylated VEGF-2 protein or variants,wherein the PEG group(s) is (are) attached via acyl or alkyl groups. Asdiscussed above, such products may be mono-pegylated or poly-pegylated(e.g., containing 2-6, and preferably 2-5, PEG groups). The PEG groupsare generally attached to the protein at the α- or ε-amino groups ofamino acids, but it is also contemplated that the PEG groups could beattached to any amino group attached to the protein, which issufficiently reactive to become attached to a PEG group under suitablereaction conditions.

The polymer molecules used in both the acylation and alkylationapproaches may be selected from among water soluble polymers asdescribed above. The polymer selected should be modified to have asingle reactive group, such as an active ester for acylation or analdehyde for alkylation, preferably, so that the degree ofpolymerization may be controlled as provided for in the present methods.An exemplary reactive PEG aldehyde is polyethylene glycolpropionaldehyde, which is water stable, or mono C1-C10 alkoxy or aryloxyderivatives thereof (see, U.S. Pat. No. 5,252,714). The polymer may bebranched or unbranched. For the acylation reactions, the polymer(s)selected should have a single reactive ester group. For the presentreductive alkylation, the polymer(s) selected should have a singlereactive aldehyde group. Generally, the water soluble polymer will notbe selected from naturally-occurring glycosyl residues since these areusually made more conveniently by mammalian recombinant expressionsystems. The polymer may be of any molecular weight, and may be branchedor unbranched.

A particularly preferred water-soluble polymer for use herein ispolyethylene glycol. As used herein, polyethylene glycol is meant toencompass any of the forms of PEG that have been used to derivatizeother proteins, such as mono-(C1-C10) alkoxy- or aryloxy-polyethyleneglycol.

In general, chemical derivatization may be performed under any suitablecondition used to react a biologically active substance with anactivated polymer molecule. Methods for preparing pegylated VEGF-2protein or variant will generally comprise the steps of (a) reacting aVEGF-2 protein or variant with polyethylene glycol (such as a reactiveester or aldehyde derivative of PEG) under conditions whereby theprotein becomes attached to one or more PEG groups, and (b) obtainingthe reaction product(s). In general, the optimal reaction conditions forthe acylation reactions will be determined case-by-case based on knownparameters and the desired result. For example, the larger the ratio ofPEG:protein, the greater the percentage of poly-pegylated product.

Reductive alkylation to produce a substantially homogeneous populationof mono-polymer/VEGF-2 protein (or variant) conjugate molecule willgenerally comprise the steps of: (a) reacting a VEGF-2 protein orvariant with a reactive PEG molecule under reductive alkylationconditions, at a pH suitable to permit selective modification of thea-amino group at the amino terminus of said VEGF-2 protein or variant;and (b) obtaining the reaction product(s).

For a substantially homogeneous population of mono-polymer/VEGF-2protein (or variant) conjugate molecules, the reductive alkylationreaction conditions are those which permit the selective attachment ofthe water soluble polymer moiety to the N-terminus of VEGF-2 protein orvariant. Such reaction conditions generally provide for pKa differencesbetween the lysine amino groups and the α-amino group at the N-terminus(the pKa being the pH at which 50% of the amino groups are protonatedand 50% are not). The pH also affects the ratio of polymer to protein tobe used. In general, if the pH is lower, a larger excess of polymer toprotein will be desired (i.e., the less reactive the N-terminal α-aminogroup, the more polymer needed to achieve optimal conditions). If the pHis higher, the polymer:protein ratio need not be as large (i.e., morereactive groups are available, so fewer polymer molecules are needed).For purposes of the present invention, the pH will generally fall withinthe range of 3-9, preferably 3-6.

Another important consideration is the molecular weight of the polymer.In general, the higher the molecular weight of the polymer, the fewerpolymer molecules may be attached to the protein. Similarly, branchingof the polymer should be taken into account when optimizing theseparameters. Generally, the higher the molecular weight (or the morebranches) the higher the polymer:protein ratio. In general, for thepegylation reactions contemplated herein, the preferred averagemolecular weight is about 2 kDa to about 100 kDa. The preferred averagemolecular weight is about 5 kDa to about 50 kDa, particularly preferablyabout 12 kDa to about 25 kDa. The ratio of water-soluble polymer toVEGF-2 protein or variant will generally range from 1:1 to 100:1,preferably (for polypegylation) 1:1 to 20:1 and (for monopegylation) 1:1to 5:1.

Using the conditions indicated above, reductive alkylation will providefor selective attachment of the polymer to any VEGF-2 protein or varianthaving an α-amino group at the amino terminus, and provide for asubstantially homogenous preparation of monopolymer/VEGF-2 protein (orvariant) conjugate. The term “monopolymer/VEGF-2 protein (or variant)conjugate” is used here to mean a composition comprised of a singlepolymer molecule attached to a molecule of VEGF-2 protein or VEGF-2variant protein. The monopolymer/VEGF-2 protein (or variant) conjugatepreferably will have a polymer molecule located at the N-terminus, butnot on lysine amino side groups. The preparation will preferably begreater than 90% monopolymer/VEGF-2 protein (or variant) conjugate, andmore preferably greater than 95% monopolymer/VEGF-2 protein (or variant)conjugate, with the remainder of observable molecules being unreacted(i.e., protein lacking the polymer moiety).

For the present reductive alkylation, the reducing agent should bestable in aqueous solution and preferably be able to reduce only theSchiff base formed in the initial process of reductive alkylation.Preferred reducing agents may be selected from sodium borohydride,sodium cyanoborohydride, dimethylamine borane, trimethylamine borane andpyridine borane. A particularly preferred reducing agent is sodiumcyanoborohydride. Other reaction parameters, such as solvent, reactiontimes, temperatures, etc., and means of purification of products, can bedetermined case-by-case based on the published information relating toderivatization of proteins with water soluble polymers (see thepublications cited herein).

Epitopes and Antibodies

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NOS:2 or 4, or an epitope of thepolypeptide sequence encoded by a polynucleotide sequence contained inATCC™ Deposit No: 97149 or 75698 or encoded by a polynucleotide thathybridizes to the complement of the sequence of SEQ ID NOS:1 or 3 orcontained in ATCC™ Deposit No: 97149 or 75698 under stringenthybridization conditions or lower stringency hybridization conditions asdefined supra. The present invention further encompasses polynucleotidesequences encoding an epitope of a polypeptide sequence of the invention(such as, for example, the sequence disclosed in SEQ ID NOS:1 or 3),polynucleotide sequences of the complementary strand of a polynucleotidesequence encoding an epitope of the invention, and polynucleotidesequences which hybridize to the complementary strand under stringenthybridization conditions or lower stringency hybridization conditionsdefined supra.

Specific monoclonal antibodies have been raised against the VEGF-2protein (SEQ ID NO:2). These monoclonal antibodies have been given thefollowing designations: 12E2; 13A2; 15C2; 13D6; 13E6; 19A3; 8G11; 11A8,15E10, 9B4, and 13G11. Monoclonal antibodies 15C2, 13D6, and 15E10 weredeposited as a group on Jun. 8, 1999, and given ATCC™ Deposit NumberPTA-198. Monoclonal antibody 13D6 was also deposited by itself on Jul.29, 1999, and given ATCC™ Deposit Number PTA-435. Monoclonal antibodies13A2, 13E6, and 9B4 were deposited as a group on Jun. 8, 1999, and givenATCC™ Deposit Number PTA-199. Monoclonal antibodies 8G11, 12E2, and13G11 were deposited as a group on Jun.8, 1999, and given ATCC™ DepositNumber PTA-200. Monoclonal antibodies 11A8 and 19A3 were deposited as agroup on Jun. 8, 1999, and given ATCC™ Deposit Number PTA-201. Theantibodies deposited in a mixture can be isolated based on theircharacteristics, such as epitope map position, affinity, species asdescribed in the Examples.

The epitopes to which the above listed monoclonal antibodies havespecificity have been mapped to the VEGF-2 protein (See FIG. 24).Furthermore, the status of each monoclonal antibody, such as therelative affinity and ELISA and Western reactivity, have been disclosedfor each of the monoclonal antibodies (See FIG. 25).

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope,” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed infra. (See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as usedherein, is defined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length. Additional non-exclusive preferred antigenicepitopes include the antigenic epitopes disclosed herein, as well asportions thereof. Antigenic epitopes are useful, for example, to raiseantibodies, including monoclonal antibodies, that specifically bind theepitope. Preferred antigenic epitopes include the antigenic epitopesdisclosed herein, as well as any combination of two, three, four, fiveor more of these antigenic epitopes. Antigenic epitopes can be used asthe target molecules in immunoassays. (See, for instance, Wilson et al.,Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg of peptide or carrier protein and Freund's adjuvant or anyother adjuvant known for stimulating an immune response. Several boosterinjections may be needed, for instance, at intervals of about two weeks,to provide a useful titer of anti-peptide antibody which can bedetected, for example, by ELISA assay using free peptide adsorbed to asolid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to other polypeptide sequences. Forexample, the polypeptides of the present invention may be fused with theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof)resulting in chimeric polypeptides. Such fusion proteins may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of anantigen across the epithelial barrier to the immune system has beendemonstrated for antigens (e.g., insulin) conjugated to an FcRn bindingpartner such as IgG or Fc fragments (see, e.g., PCT Publications WO96/22024 and WO 99/04813). IgG Fusion proteins that have adisulfide-linked dimeric structure due to the IgG portion desulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:1 or 3 and the polypeptides encoded by these polynucleotides may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

Antibodies

Further polypeptides of the invention relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,polypeptide fragment, or variant of SEQ ID NOS:2 or 4, and/or anepitope, of the present invention (as determined by immunoassays wellknown in the art for assaying specific antibody-antigen binding).Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Inmunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Preferred epitopes of theinvention include: Leu-37 to about Glu-45 in SEQ ID NO:2, from aboutTyr-58 to about Gly-66 in SEQ ID NO:2, from about Gln-73 to about Glu-81in SEQ ID NO:2, from about Asp-100 to about Cys-108 in SEQ ID NO:2, fromabout Gly-140 to about Leu-148 in SEQ ID NO:2, from about Pro-168 toabout Val-176 in SEQ ID NO:2, from about His-183 to about Lys-191 in SEQID NO:2, from about Ile-201 to about Thr-209 in SEQ ID NO:2, from aboutAla-216 to about Tyr-224 in SEQ ID NO:2, from about Asp-244 to aboutHis-254 in SEQ ID NO:2, from about Gly-258 to about Glu-266 in SEQ IDNO:2, from about Cys-272 to about Ser-280 in SEQ ID NO:2, from aboutPro-283 to about Ser-291 in SEQ ID NO:2, from about Cys-296 to aboutGln-304 in SEQ ID NO:2, from about Ala-307 to about Cys-316 in SEQ IDNO:2, from about Val-319 to about Cys-335 in SEQ ID NO:2, from aboutCys-339 to about Leu-347 in SEQ ID NO:2, from about Cys-360 to aboutGlu-373 in SEQ ID NO:2, from about Tyr-378 to about Val-386 in SEQ IDNO:2, and from about Ser-388 to about Ser-396 in SEQ ID NO:2, as well aspolynucleotides that encode these epitopes. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, rat and/or rabbit homologs of human proteinsand the corresponding epitopes thereof. Antibodies that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M,5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, ¹⁰⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. Preferably, antibodies of the present invention bind an antigenicepitope disclosed herein, or a portion thereof. The invention featuresboth receptor-specific antibodies and ligand-specific antibodies. Theinvention also features receptor-specific antibodies which do notprevent ligand binding but prevent receptor activation. Receptoractivation (i.e., signaling) may be determined by techniques describedherein or otherwise known in the art. For example, receptor activationcan be determined by detecting the phosphorylation (e.g., tyrosine orserine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra). In specific embodiments, antibodies are provided thatinhibit ligand activity or receptor activity by at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples. In a non-limiting example, mice canbe immunized with a polypeptide of the invention or a cell expressingsuch peptide. Once an immune response is detected, e.g., antibodiesspecific for the antigen are detected in the mouse serum, the mousespleen is harvested and splenocytes isolated. The splenocytes are thenfused by well known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC™. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entirety. Humanized antibodies are antibodymolecules from non-human species antibody that binds the desired antigenhaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework regions from a human immunoglobulinmolecule. Often, framework residues in the human framework regions willbe substituted with the corresponding residue from the CDR donorantibody to alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NOS:2 or 4.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa califomica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

As discussed, supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of SEQ ID NOS:2 or 4 may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. Further, the polypeptides corresponding to SEQ ID NOS:2 or 4may be fused or conjugated to the above antibody portions to facilitatepurification. One reported example describes chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature331:84-86 (1988). The polypeptides of the present invention fused orconjugated to an antibody having disulfide-linked dimeric structures(due to the IgG) may also be more efficient in binding and neutralizingother molecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In manycases, the Fc part in a fusion protein is beneficial in therapy anddiagnosis, and thus can result in, for example, improved pharmacokineticproperties. (EP A 232,262). Alternatively, deleting the Fc part afterthe fusion protein has been expressed, detected, and purified, would bedesired. For example, the Fc portion may hinder therapy and diagnosis ifthe fusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hIL-5, have been fusedwith Fc portions for the purpose of high-throughput screening assays toidentify antagonists of hIL-5. (See, Bennett et al., J. MolecularRecognition 8:52-58 (1995); Johanson et al., J. Biol. Chem.270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 1251, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the geneof the present invention may be useful as a cell specific marker, ormore specifically as a cellular marker that is differentially expressedat various stages of differentiation and/or maturation of particularcell types. Monoclonal antibodies directed against a specific epitope,or combination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Assays For Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffuisionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., 3H or 125I)in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10^(−12 M,) 5×10⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291-302(1994), which describes the use of a retroviral vector to deliver themdr1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide ofinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99 mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or calorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

Any VEGF-2 polypeptide can be used to generate fusion proteins. Forexample, the VEGF-2 polypeptide, when fused to a second protein, can beused as an antigenic tag. Antibodies raised against the VEGF-2polypeptide can be used to indirectly detect the second protein bybinding to the VEGF-2. Moreover, because secreted proteins targetcellular locations based on trafficking signals, the VEGF-2 polypeptidescan be used as a targeting molecule once fused to other proteins.

Examples of domains that can be fused to VEGF-2 polypeptides include notonly heterologous signal sequences, but also other heterologousfunctional regions. The fusion does not necessarily need to be direct,but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the VEGF-2 polypeptide. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the VEGF-2 polypeptide to improve stability andpersistence during purification from the host cell or subsequenthandling and storage. Also, peptide moieties may be added to the VEGF-2polypeptide to facilitate purification. Such regions may be removedprior to final preparation of the VEGF-2 polypeptide. The addition ofpeptide moieties to facilitate handling of polypeptides are familiar androutine techniques in the art.

Moreover, VEGF-2 polypeptides, including fragments, and specificallyepitopes, can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. One reported example describes chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86(1988).) Fusion proteins having disulfide-linked dimeric structures (dueto the IgG) can also be more efficient in binding and neutralizing othermolecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the VEGF-2 polypeptides can be fused to marker sequences, suchas a peptide which facilitates purification of VEGF-2. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the “HA” tag, corresponds to an epitope derivedfrom the influenza hemagglutinin protein. (Wilson et al., Cell 37:767(1984).)

Thus, any of these above fusions can be engineered using the VEGF-2polynucleotides or the polypeptides.

Vectors and Host Cells

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofVEGF-2 polypeptides or peptides by recombinant techniques.

Host cells are genetically engineered (transduced, transformed, ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants, or amplifying the VEGF-2 genes of the invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide sequence may be included in any one of a variety ofexpression vectors for expressing a polypeptide. Such vectors includechromosomal, nonchromosomal and synthetic DNA sequences, e.g.,derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids;vectors derived from combinations of plasmids and phage DNA, viral DNAsuch as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However,any other plasmid or vector may be used so long as it is replicable andviable in the host.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain at least oneselectable marker gene to provide a phenotypic trait for selection oftransformed host cells. Such markers include dihydrofolate reductase(DHFR) or neomycin resistance for eukaryotic cell culture, andtetracycline or ampicillin resistance for culturing in E. coli and otherbacteria.

The vector containing the appropriate DNA sequence as herein abovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein. Representative examples of appropriate hosts,include but are not limited to: bacterial cells, such as E. coli,Salmonella typhimurium, and Streptomyces; fungal cells, such as yeast;insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cellssuch as CHO, COS, and Bowes melanoma; and plant cells. The selection ofan appropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example—bacterial: pQE70, pQE60 and pQE-9,available from Qiagen; pBS vectors, Phagescript vectors, Bluescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; andptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 andpSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia. Other suitable vectors will be readily apparent to theskilled artisan.

In addition to the use of expression vectors in the practice of thepresent invention, the present invention further includes novelexpression vectors comprising operator and promoter elements operativelylinked to nucleotide sequences encoding a protein of interest. Oneexample of such a vector is pHE4a which is described in detail below.

As summarized in FIGS. 16 and 17, components of the pHE4a vector (SEQ IDNO:9) include: 1) a neomycinphosphotransferase gene as a selectionmarker, 2) an E. coli origin of replication, 3) a T5 phage promotersequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence,6) the lactose operon repressor gene (lacIq) and 7) a multiple cloningsite linker region. The origin of replication (oriC) is derived frompUC19 (LTI, Gaithersburg, Md.). The promoter sequence and operatorsequences were made synthetically. Synthetic production of nucleic acidsequences is well known in the art. CLONTECH 95/96 Catalog, pages215-216, CLONTECH, 1020 East Meadow Circle, Palo Alto, Calif. 94303. ThepHE4a vector was deposited with the ATCC™ on Feb. 25, 1998, and givenaccession number 209645.

A nucleotide sequence encoding VEGF-2 (SEQ ID NO:1), is operativelylinked to the promoter and operator of pHE4a by restricting the vectorwith NdeI and either XbaI, BamHI, XhoI, or Asp718, and isolating thelarger fragment (the multiple cloning site region is about 310nucleotides) on a gel. The nucleotide sequence encoding VEGF-2 (SEQ IDNO:1) having the appropriate restriction sites is generated, forexample, according to the PCR protocol described in Example 1, using PCRprimers having restriction sites for NdeI (as the 5′ primer) and eitherXbaI, BaniHI, XhoI, or Asp718 (as the 3′ primer). The PCR insert is gelpurified and restricted with compatible enzymes. The insert and vectorare ligated according to standard protocols.

As noted above, the pHE4a vector contains a lacIq gene. LacIq is anallele of the lacI gene which confers tight regulation of the lacoperator. Amann, E. et al., Gene 69:301-315 (1988); Stark, M., Gene51:255-267 (1987). The lacIq gene encodes a repressor protein whichbinds to lac operator sequences and blocks transcription of down-stream(i.e., 3′) sequences. However, the lacIq gene product dissociates fromthe lac operator in the presence of either lactose or certain lactoseanalogs, e.g., isopropyl B-D-thiogalactopyranoside (IPTG). VEGF-2 thusis not produced in appreciable quantities in uninduced host cellscontaining the pHE4a vector. Induction of these host cells by theaddition of an agent such as IPTG, however, results in the expression ofthe VEGF-2 coding sequence.

The promoter/operator sequences of the pHE4a vector (SEQ ID NO:10)comprise a T5 phage promoter and two lac operator sequences. Oneoperator is located 5′ to the transcriptional start site and the otheris located 3′ to the same site. These operators, when present incombination with the LacIq gene product, confer tight repression ofdown-stream sequences in the absence of a lac operon inducer, e.g.,IPTG. Expression of operatively linked sequences located down-streamfrom the lac operators may be induced by the addition of a lac operoninducer, such as IPTG. Binding of a lac inducer to the lacIq proteinsresults in their release from the lac operator sequences and theinitiation of transcription of operatively linked sequences. Lac operonregulation of gene expression is reviewed in Devlin, T., TEXTBOOK OFBIOCHEMISTRY WITH CLINICAL CORRELATIONS, 4th Edition (1997), pages802-807.

The pHE4 series of vectors contain all of the components of the pHE4avector except for the VEGF-2 coding sequence. Features of the pHE4avectors include optimized synthetic T5 phage promoter, lac operator, andShine-Delagamo sequences. Further, these sequences are also optimallyspaced so that expression of an inserted gene may be tightly regulatedand high level of expression occurs upon induction.

Among known bacterial promoters suitable for use in the production ofproteins of the present invention include the E. coli lacI and lacZpromoters, the T3 and T7 promoters, the gpt promoter, the lambda PR andPL promoters and the trp promoter. Suitable eukaryotic promoters includethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous Sarcoma Virus (RSV), and metallothionein promoters,such as the mouse metallothionein-I promoter.

The pHE4a vector also contains a Shine-Delgarno sequence 5′ to the AUGinitiation codon. Shine-Delgamo sequences are short sequences generallylocated about 10 nucleotides up-stream (i.e., 5′) from the AUGinitiation codon. These sequences essentially direct prokaryoticribosomes to the AUG initiation codon. Thus, the present invention isalso directed to expression vector useful for the production of theproteins of the present invention. This aspect of the invention isexemplified by the pHE4a vector (SEQ ID NO:9).

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art. In a furtherembodiment, the present invention relates to host cells containing theabove-described construct. The host cell can be a higher eukaryoticcell, such as a mammalian cell, or a lower eukaryotic cell, such as ayeast cell, or the host cell can be a prokaryotic cell, such as abacterial cell. Introduction of the construct into the host cell can beeffected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, electroporation, transduction, infection, or other methods(Davis, L., et al., Basic Methods in Molecular Biology (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook. et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1989), the disclosure of which is herebyincorporated by reference.

Transcription of a DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100 to 270), a cytomegalovirus early promoterenhancer, a polyoma enhancer on the late side of the replication origin,and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC™ 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification. Microbial cells employed in expression of proteins can bedisrupted by any convenient method, well known to those skilled in theart, including freeze-thaw cycling, sonication, mechanical disruption,or use of cell lysing agents.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., VEGF-2 sequence), and/or to include geneticmaterial (e.g., heterologous promoters) that is operably associated withVEGF-2 sequence of the invention, and which activates, alters, and/oramplifies endogenous VEGF-2 polynucleotides. For example, techniquesknown in the art may be used to operably associate heterologous controlregions and endogenous polynucleotide sequences (e.g. encoding VEGF-2)via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issuedJun. 24, 1997; International Publication No. WO 96/29411, published Sep.26, 1996; International Publication No. WO 94/12650, published Aug. 4,1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); andZijlstra et al., Nature 342:435-438 (1989), the disclosures of each ofwhich are incorporated by reference in their entireties).

The host cell can be a higher eukaryotic cell, such as a mammalian cell(e.g., a human derived cell), or a lower eukaryotic cell, such as ayeast cell, or the host cell can be a prokaryotic cell, such as abacterial cell. The host strain may be chosen which modulates theexpression of the inserted gene sequences, or modifies and processes thegene product in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thusexpression of the genetically engineered polypeptide may be controlled.Furthermore, different host cells have characteristics and specificmechanisms for the translational and post-translational processing andmodification (e.g., glycosylation, phosphorylation, cleavage) ofproteins. Appropriate cell lines can be chosen to ensure the desiredmodifications and processing of the protein expressed.

The polypeptides can be recovered and purified from recombinant cellcultures by methods used heretofore, including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. It is preferred to have low concentrations(approximately 0.1-5 mM) of calcium ion present during purification(Price et al., J. Biol. Chem. 244:917 (1969)). Protein refolding stepscan be used, as necessary, in completing configuration of the matureprotein. Finally, high performance liquid chromatography (HPLC) can beemployed for final purification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated with mammalian or other eukaryotic carbohydrates or may benon-glycosylated. Polypeptides of the invention may also include aninitial methionine amino acid residue.

VEGF-2 Agonist and Antagonists

This invention is also related to a method of screening compounds toidentify those which are VEGF-2 agonists or antagonists. An example ofsuch a method takes advantage of the ability of VEGF-2 to significantlystimulate the proliferation of human endothelial cells in the presenceof the comitogen Con A. Endothelial cells are obtained and cultured in96-well flat-bottomed culture plates (Costar, Cambridge, Mass.) in areaction mixture supplemented with Con-A (Calbiochem, La Jolla, Calif.).Con-A, polypeptides of the present invention and the compound to bescreened are added. After incubation at 37EC, cultures are pulsed with 1FCi of ³[H]thymidine (5 Ci/mmol; 1 Ci=37 BGq; NEN) for a sufficient timeto incorporate the ³[H] and harvested onto glass fiber filters(Cambridge Technology, Watertown, Mass.). Mean ³[H]-thymidineincorporation (cpm) of triplicate cultures is determined using a liquidscintillation counter (Beckman Instruments, Irvine, Calif.). Significant³[H]thymidine incorporation, as compared to a control assay where thecompound is excluded, indicates stimulation of endothelial cellproliferation.

To assay for antagonists, the assay described above is performed and theability of the compound to inhibit ³[H]thymidine incorporation in thepresence of VEGF-2 indicates that the compound is an antagonist toVEGF-2. Alternatively, VEGF-2 antagonists may be detected by combiningVEGF-2 and a potential antagonist with membrane-bound VEGF-2 receptorsor recombinant receptors under appropriate conditions for a competitiveinhibition assay. VEGF-2 can be labeled, such as by radioactivity, suchthat the number of VEGF-2 molecules bound to the receptor can determinethe effectiveness of the potential antagonist.

Alternatively, the response of a known second messenger system followinginteraction of VEGF-2 and receptor would be measured and compared in thepresence or absence of the compound. Such second messenger systemsinclude but are not limited to, cAMP guanylate cyclase, ion channels orphosphoinositide hydrolysis. In another method, a mammalian cell ormembrane preparation expressing the VEGF-2 receptor is incubated withlabeled VEGF-2 in the presence of the compound. The ability of thecompound to enhance or block this interaction could then be measured.

Potential VEGF-2 antagonists include an antibody, or in some cases, anoligonucleotide, which bind to the polypeptide and effectively eliminateVEGF-2 function. Alternatively, a potential antagonist may be a closelyrelated protein which binds to VEGF-2 receptors, however, they areinactive forms of the polypeptide and thereby prevent the action ofVEGF-2. Examples of these antagonists include a negative dominant mutantof the VEGF-2 polypeptide, for example, one chain of the hetero-dimericform of VEGF-2 may be dominant and may be mutated such that biologicalactivity is not retained. An example of a negative dominant mutantincludes truncated versions of a dimeric VEGF-2 which is capable ofinteracting with another dimer to form wild type VEGF-2, however, theresulting homo-dimer is inactive and fails to exhibit characteristicVEGF activity.

Another potential VEGF-2 antagonist is an antisense construct preparedusing antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, the 5′ coding portion of the polynucleotide sequence,which encodes for the mature polypeptides of the present invention, isused to design an antisense RNA oligonucleotide of from about 10 to 40base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix—see Lee et al., Nucl. Acids Res.6:3073 (1979); Cooney et al.,Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)),thereby preventing transcription and the production of VEGF-2. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the VEGF-2 polypeptide(Antisense-Okano, J. Neurochem.56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of VEGF-2. Potential VEGF-2 antagonists also includesmall molecules which bind to and occupy the active site of thepolypeptide thereby making the catalytic site inaccessible to substratesuch that normal biological activity is prevented. Examples of smallmolecules include but are not limited to small peptides or peptide-likemolecules.

Antisense oligonucleotide technology provides a novel approach to theinhibition of gene expression (see generally, Agrawal (1992) Trends inBiotech. 10:152; Wagner (1994) Nature 372:333-335; and Stein et al.(1993) Science 261:1004-1012). By binding to the complementary nucleicacid sequence (the sense strand), antisense oligonucleotide are able toinhibit splicing and translation of RNA. In this way, antisenseoligonucleotides are able to inhibit protein expression. Antisenseoligonucleotides have also been shown to bind to genomic DNA, forming atriplex, and inhibit transcription. Furthermore, a 17 mer base sequencestatistically occurs only once in the human genome, and thus extremelyprecise targeting of specific sequences is possible with such antisenseoligonucleotides.

The antagonists may be employed to limit angiogenesis necessary forsolid tumor metastasis. The identification of VEGF-2 can be used for thegeneration of certain inhibitors of vascular endothelial growth factor.Since angiogenesis and neovascularization are essential steps in solidtumor growth, inhibition of angiogenic activity of the vascularendothelial growth factor is very useful to prevent the further growth,retard, or even regress solid tumors. Although the level of expressionof VEGF-2 is extremely low in normal tissues including breast, it can befound expressed at moderate levels in at least two breast tumor celllines that are derived from malignant tumors. It is, therefore, possiblethat VEGF-2 is involved in tumor angiogenesis and growth.

Gliomas are also a type of neoplasia which may be treated with theantagonists of the present invention.

The antagonists may also be used to treat chronic inflammation caused byincreased vascular permeability. In addition to these disorders, theantagonists may also be employed to treat retinopathy associated withdiabetes, rheumatoid arthritis and psoriasis.

The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinafter described.

Truncated versions of VEGF2 can also be produced that are capable ofinteracting with wild type VEGF2 to form dimers that fail to activateendothelial cell growth, therefore inactivating the endogenous VEGF2.Or, mutant forms of VEGF2 form dimers themselves and occupy the ligandbinding domain of the proper tyrosine kinase receptors on the targetcell surface, but fail to activate cell growth.

Alternatively, antagonists to the polypeptides of the present inventionmay be employed which bind to the receptors to which a polypeptide ofthe present invention normally binds. The antagonists may be closelyrelated proteins such that they recognize and bind to the receptor sitesof the natural protein, however, they are inactive forms of the naturalprotein and thereby prevent the action of VEGF2 since receptor sites areoccupied. In these ways, the action of the VEGF2 is prevented and theantagonist/inhibitors may be used therapeutically as an anti-tumor drugby occupying the receptor sites of tumors which are recognized by VEGF2or by inactivating VEGF2 itself. The antagonist/inhibitors may also beused to prevent inflammation due to the increased vascular permeabilityaction of VEGF2. The antagonist/inhibitors may also be used to treatsolid tumor growth, diabetic retinopathy, psoriasis and rheumatoidarthritis.

The antagonist/inhibitors may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinabove described.Moreover, as shown in Example 9, antibodies specific for VEGF-2 may becombined with VEGF-2 polypeptides to increase endothelial cell response.Endothelial cells responding to a combination of VEGF-2 polypeptide andVEGF-2 specific antibodies include vascular or lymphatic vessels. Thecombination of VEGF-2 specific antibodies and VEGF-2 polypeptide may beused to treat individuals in need of an increase in proliferation ofendothelial cells, such as angiogenesis and/or lymphangiogenesis, asdescribed throughout the specification.

Therapeutic Applications of VEGF-2

As used in the section below, “VEGF-2” is intended to refer to thefull-length and mature forms of VEGF-2 polynucleotides and polypeptidesdescribed herein and to the VEGF-2 analogs, derivatives, and mutantpolynucleotides and polypeptides described herein.

The VEGF-2 polypeptide of the present invention is a mitogen forphotoreceptor cells. As shown in FIGS. 12-15, VEGF-2 increases cellnumber, cell survival, rhodopsin expression, and the number of rhodopsincells in retinal cultures.

Accordingly, VEGF-2 may be employed to treat disorders of the eye,including injuries and diseases. These disorders include angioidstreaks, retinitis pigmentosa, Keam's Syndrome, pigment patterndystrophies, retinal perforations, retinitis, chorioretinitis,cytomegalovirus retinitis, acute retinal necrosis syndrome, centralalveolar choroidal dystrophy, dominant drusen, hereditary hemorrhagicmacular dystrophy, North Carolina macular dystrophy, pericentralchoroidal dystrophy, adult foveomacular dystrophy, benign concentricannular macular dystrophy, central aureolar pigment epithelialdystrophy, congenital macular coloboma, dominantly inherited cystoidmacular edema, familial foveal retinoschisis, fenestrated sheen maculardystrophy, progressive foveal dystrophy, slowly progressive maculardystrophy, Sorsby's pseudoinflammatory dystrophy, cone-rod dystrophy,progressive cone dystrophy, Leber's congenital amaurosis, Goldman-Favresyndrome, Bardet-Biedl syndrome, Bassen-Komzweig syndrome(abetalipoproteinemia), Best disease (vitelliform dystrophy),choroidemia, gyrate atrophy, congenital amaurosis, Refsum syndrome,Stargardt disease and Usher syndrome. Other retinopathies that maybenefit from VEGF-2 administration include age-related maculardegeneration (dry and wet forms), diabetic retinopathy, peripheralvitreoretinopathies, photic retinopathies, surgery-inducedretinopathies, viral retinopathies (such as HIV retinopathy related toAIDS), ischemic retinopathies, retinal detachment and traumaticretinopathy.

VEGF-2 may be administered along with other proteins which aretherapeutic for eye cells, including, but not limited to: retinoic acid,mitogens such as insulin, insulin-like growth factors, epidermal growthfactor, vasoactive growth factor, pituitary adenylate cyclase activatingpolypeptide and somatostatin; neurotrophic factors such as glial cellline-derived neurotrophic factor, brain derived neurotrophic factor,neurotrophin-3, neurotrophin-4/5, neurotrophin-6, insulin-like growthfactor, ciliary neurotrophic factor, acidic and basic fibroblast growthfactors, fibroblast growth factor-5, transforming growth factor-beta,and cocaine-amphetamine regulated transcript (CART); and other growthfactors such as epidermal growth factor, leukemia inhibitory factor,interleukins, interferons, and colony stimulating factors; as well asmolecules and materials which are the functional equivalents to thesefactors.

Additionally, antibodies may further be used in an immunoassay to detectthe presence of tumors in certain individuals. Enzyme immunoassay can beperformed from the blood sample of an individual. Elevated levels ofVEGF2 can be considered diagnostic of cancer.

Pharmaceutical Compositions

The VEGF-2 polypeptides and polynucleotides of the present invention maybe employed in combination with a suitable pharmaceutical carrier tocomprise a pharmaceutical composition. Such compositions comprise atherapeutically effective amount of the polypeptide, polynucleotide,agonist or antagonist and a pharmaceutically acceptable carrier orexcipient. Such a carrier includes, but is not limited to, antioxidants,preservatives, coloring, flavoring and diluting agents, emulsifyingagents, suspending agents, solvents, fillers, bulking agents, buffers,delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants.The formulation should suit the mode of administration. For example,suitable vehicles include saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof.

The primary solvent in a vehicle may be either aqueous or non-aqueous innature. In addition, the vehicle may contain otherpharmaceutically-acceptable excipients for modifying or maintaining thepH, osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution, or odor of the formulation. Similarly, the vehicle maycontain still other pharmaceutically-acceptable excipients for modifyingor maintaining the rate of release of VEGF-2, or for promoting theabsorption or penetration of VEGF-2 across the membranes of the eye.Such excipients are those substances usually and customarily employed toformulate dosages for parenteral administration in either unit dose ormulti-dose form.

Once the therapeutic composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. Such formulations may be stored eitherin a ready to use form or in a form, e.g., lyophilized, requiringreconstitution prior to administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainers can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides, agonists and antagonists of the present invention may beemployed in conjunction with other therapeutic compounds.

The VEGF-2 polypeptide or polynucleotide may be administered inpharmaceutical compositions in combination with one or morepharmaceutically acceptable excipients. It will be understood that, whenadministered to a human patient, the total daily usage of thepharmaceutical compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular patientwill depend upon a variety of factors including the type and degree ofthe response to be achieved; the specific composition an other agent, ifany, employed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the composition; the duration of the treatment; drugs(such as a chemotherapeutic agent) used in combination or coincidentalwith the specific composition; and like factors well known in themedical arts. Suitable formulations, known in the art, can be found inRemington's Pharmaceutical Sciences (latest edition), Mack PublishingCompany, Easton, Pa.

The VEGF-2 composition to be used in the therapy will be formulated anddosed in a fashion consistent with good medical practice, taking intoaccount the clinical condition of the individual patient (especially theside effects of treatment with VEGF-2 alone), the site of delivery ofthe VEGF-2 composition, the method of administration, the scheduling ofadministration, and other factors known to practitioners. The “effectiveamount” of VEGF-2 for purposes herein is thus determined by suchconsiderations.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, intraarticular, subcutaneous, intranasal, intratracheal,intraocular or intradermal routes. The pharmaceutical compositions areadministered in an amount which is effective for treating and/orprophylaxis of the specific indication. In most cases, the VEGF-2 dosageis from about 1 μg/kg to about 30 mg/kg body weight daily, taking intoaccount the routes of administration, symptoms, etc. However, the dosagecan be as low as 0.001 μg/kg. For example, in the specific case oftopical administration dosages are preferably administered from about0.01 μg to 9 mg per cm². In the case of intraocular administration,dosages are preferably administered from about 0.001 μg/ml to about 10mg/ml, and more preferably from about 0.05 mg/ml to about 4 mg/ml.

As a general proposition, the total pharmaceutically effective amount ofthe VEGF-2 administered parenterally per more preferably dose will be inthe range of about 1 μg/kg/day to 100 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. If given continuously, the VEGF-2 is typically administeredat a dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by1-4 injections per day or by continuous subcutaneous infusions, forexample, using a mini-pump. An intravenous bag solution or bottlesolution may also be employed.

VEGF-2 is also suitably administered by sustained-release systems.Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (U. Sidman et al., Biopolymers 22:547-556(1983)), poly(2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed.Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105(1982)), ethylene vinyl acetate (R. Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-release VEGF-2compositions also include liposomally entrapped VEGF-2. Liposomescontaining VEGF-2 are prepared by methods known per se: DE 3,218,121;Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688-3692 (1985); Hwanget al., Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980); EP 52,322; EP36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal VEGF-2 therapy.

For parenteral administration, in one embodiment, the VEGF-2 isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

In certain embodiments, VEGF-2 is administered orally. VEGF-2 which isadministered in this fashion may be encapsulated and may be formulatedwith or without those carriers customarily used in the compounding ofsolid dosage forms. The capsule may be designed to release the activeportion of the formulation at the point in the gastrointestinal tractwhen bioavailability is maximized and pre-systemic degradation isminimized. Additional excipients may be included to facilitateabsorption of VEGF-2. Diluents, flavorings, low melting point waxes,vegetable oils, lubricants, suspending agents, tablet disintegratingagents, and binders may also be employed.

VEGF-2 may also be administered to the eye to treat photoreceptorinjuries, disorders and pathologies in animals and humans as a drop, orwithin ointments, gels, liposomes, microparticulates, or biocompatiblepolymer discs, pellets or carried within contact lenses. The intraocularcomposition may also contain a physiologically compatible ophthalmicvehicle as those skilled in the art can select using conventionalcriteria. The vehicles may be selected from the known ophthalmicvehicles which include but are not limited to water, polyethers such aspolyethylene glycol 400, polyvinyls such as polyvinyl alcohol, povidone,cellulose derivatives such as carboxymethylcellulose, methylcelluloseand hydroxypropyl methylcellulose, petroleum derivatives such as mineraloil and white petrolatum, animal fats such as lanolin, vegetable fatssuch as peanut oil, polymers of acrylic acid such ascarboxylpolymethylene gel, polysaccharides such as dextrans andglycosaminoglycans such as sodium chloride and potassium, chloride, zincchloride and buffer such as sodium bicarbonate or sodium lactate. Highmolecular weight molecules can also be used. Physiologically compatiblepreservatives which do not inactivate the VEGF-2 present in thecomposition include alcohols such as chlorobutanol, benzalknoniumchloride and EDTA, or any other appropriate preservative known to thoseskilled in the art.

For example, VEGF-2 may be administered directly intraocularly fromabout 1 μg/eye to about 1 mg/eye in a single injection or in multipleinjections. The formulation of topical ophthalmic preparations,including ophthalmic solutions, suspensions and ointments is well knownto those skilled in the art (see Remington's Pharmaceutical Sciences,18th Edition, Chapter 86, pages 1581-1592, Mack Publishing Company,1990). Other modes of administration are available, includingintracameral injections (which may be made directly into the anteriorchamber or directly into the vitreous chamber), subconjunctivalinjections and retrobulbar injections, and methods and means forproducing ophthalmic preparations suitable for such modes ofadministration are also well known. VEGF-2 may also be administered inthe subretinal space between the photoreceptor layer and retinalpigmentosa epithelial layers.

As used herein, “extraocular” refers to the ocular surface and the(external) space between the eyeball and the eyelid. Examples ofextraocular regions include the eyelid fomix or cul-de-sac, theconjunctival surface and the corneal surface. This location is externalto all ocular tissue and an invasive procedure is not required to accessthis region. Examples of extraocular systems include inserts and“topically” applied drops, gels or ointments which may be used todeliver therapeutic material to these regions. Extraocular devices aregenerally easily removable, even by the patient.

The following patents disclose extraocular systems which are used toadminister drugs to the extraocular regions. Higuchi et al. disclose inU.S. Pat. Nos. 3,981,303, 3,986,510 and 3,995,635 a biodegradable ocularinsert which contains a drug. The insert can be made in different shapesfor retention in the cul-de-sac of the eyeball, the extraocular spacebetween the eyeball and the eyelid. Several common biocompatiblepolymers are disclosed as suitable for use in fabricating this device.These polymers include zinc alginate, poly(lactic acid), poly(vinylalcohol), poly(anhydrides) and poly(glycolic acid). The patents alsodescribe membrane coated devices with reduced permeation to the drug andhollow chambers holding the drug formulation.

U.S. Pat. No. 4,217,898, discloses microporous reservoirs which are usedfor controlled drug delivery. These devices are placed extraocularly inthe ocular cul-de-sac. Among the polymer systems of interest arepoly(vinylchloride)-co-poly(vinyl acetate)copolymers. Kaufinan disclosesin U.S. Pat. Nos. 4,865,846 and 4,882,150 an ophthalmic drug deliverysystem which contains at least one bio-erodible material or ointmentcarrier for the conjunctival sac. The patent discloses polymer systems,such as poly(lactide), poly(glycolide), poly(vinyl alcohol) and crosslinked collagen as suitable delivery systems.

In the presently described use of VEGF-2 of the treatment of retinaldisease or injury it is also advantageous that a topically appliedophthalmic formulation include an agent to promote the penetration ortransport of the therapeutic agent into the eye. Such agents are knownin the art. For example, Ke et al., U.S. Pat. No. 5,221,696 disclose theuse of materials to enhance the penetration of ophthalmic preparationsthrough the cornea.

Intraocular systems are those systems which are suitable for use in anytissue compartment within, between or around the tissue layers of theeye itself. These locations include subconjunctival (under the ocularmucous membrane adjacent to the eyeball), orbital (behind the eyeball),and intracameral (within the chambers of the eyeball itself). Incontrast to extraocular systems, an invasive procedure consisting ofinjection or implantation is required to access these regions.

The following patents disclose intraocular devices. Wong, U.S. Pat. No.4,853,224, discloses microencapsulated drugs for introduction into thechamber of the eye. Polymers which are used in this system includepolyesters and polyethers. Lee, U.S. Pat. No. 4,863,457, discloses abiodegradable device which is surgically implanted intraocularly for thesustained release of therapeutic agents. The device is designed forsurgical implantation under the conjunctiva (mucous membrane of theeyeball). Krezancaki, U.S. Pat. No. 4,188,373, discloses apharmaceutical vehicle which gels at human body temperature. Thisvehicle is an aqueous suspension of the drug and gums or cellulosederived synthetic derivatives. Haslam et al. disclose in U.S. Pat. Nos.4,474,751 and 4,474,752 a polymer-drug system which is liquid at roomtemperature and gels at body temperature. Suitable polymers used in thissystem include polyoxyethylene and polyoxypropylene. Davis et al.disclose in U.S. Pat. No. 5,384,333 a biodegradable injectable drugdelivery polymer which provides long term drug release. The drugcomposition is made up of a pharmaceutically active agent in abiodegradable polymer matrix, where the polymer matrix is a solid attemperatures in the range 20 EC to 37 EC, and is flowable attemperatures in the range 38 EC to 52 EC. The drug delivery polymer isnot limited to the delivery of soluble or liquid drug formulations. Forexample, the polymer can be used as a matrix for stabilizing andretaining at the site of injection drug-containing microspheres,liposomes or other particulate-bound drugs.

A particularly suitable vehicle for intraocular injection is steriledistilled water in which VEGF-2 is formulated as a sterile, isotonicsolution, properly preserved. Yet another ophthalmic preparation mayinvolve the formulation of VEGF-2 with an agent, such as injectablemicrospheres or liposomes, that provides for the slow or sustainedrelease of the protein which may then be delivered as a depot injection.Other suitable means for the intraocular introduction of VEGF-2 includesimplantable drug delivery devices which contain VEGF-2.

The ophthalmic preparations of the present invention, particularlytopical preparations, may include other components, for exampleophthalmically acceptable preservatives, tonicity agents, cosolvents,wetting agents, complexing agents, buffering agents, antimicrobials,antioxidants and surfactants, as are well known in the art. For example,suitable tonicity enhancing agents include alkali metal halides(preferably sodium or potassium chloride), mannitol, sorbitol and thelike. Sufficient tonicity enhancing agent is advantageously added sothat the formulation to be instilled into the eye is hypotonic orsubstantially isotonic. Suitable preservatives include, but are notlimited to, benzalkonium chloride, thimerosal, phenethyl alcohol,methylparaben, propylparaben, chlorhexidine, sorbic acid and the like.Hydrogen peroxide may also be used as preservative. Suitable cosolventsinclude, but are not limited to, glycerin, propylene glycol andpolyethylene glycol. Suitable complexing agents include caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude, but are not limited to, sorbitan esters, polysorbates such aspolysorbate 80, tromethamine, lecithin, cholesterol, tyloxapol and thelike. The buffers can be conventional buffers such as borate, citrate,phosphate, bicarbonate, or Tris-HCl.

The formulation components are present in concentration that areacceptable to the extraocular or intraocular site of administration. Forexample, buffers are used to maintain the composition at physiologicalpH or at slightly lower pH, typically within a pH range of from about 5to about 8.

Additional formulation components may include materials which providefor the prolonged ocular residence of the extraocularly administeredtherapeutic agent so as to maximize the topical contact and promoteabsorbtion. Suitable materials include polymers or gel forming materialswhich provide for increased viscosity of the ophthalmic preparation.Chitosan is a particularly suitable material as an ocular release-ratecontrolling agent in sustained release liquid ophthalmic drugformulations (see U.S. Pat. No. 5,422,116, Yen, et. al.) The suitabilityof the formulations of the instant invention for controlled release(e.g., sustained and prolonged delivery) of an ophthalmic treating agentin the eye can be determined by various procedures known in the art,e.g., as described in Journal of Controlled Release 6:367-373, 1987, aswell as variations thereof.

Yet another ophthalmic preparation may involve an effective quantity ofVEGF-2 in a mixture with non-toxic ophthalmically acceptable excipientswhich are suitable for the manufacture of tablets. By dissolving thetablets in sterile water, or other appropriate vehicle, ophthalmicsolutions can be prepared in unit dose form. Suitable excipientsinclude, but are not limited to, inert diluents, such as calciumcarbonate, sodium carbonate or bicarbonate, lactose, or calciumphosphate; or binding agents, such as starch, gelatin, or acacia; orlubricating agents such as magnesium stearate, stearic acid, or talc.

Generally, the formulations are prepared by contacting the VEGF-2uniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes. Suitable formulations, known in the art, can be found inRemington's Pharmaceutical Sciences (latest edition), Mack PublishingCompany, Easton, Pa.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

VEGF-2 is typically formulated in such vehicles at a concentration ofabout 0.01 μg/ml to 100 mg/ml, preferably 0.01 μg/ml to 10 mg/ml, at apH of about 3 to 8. It will be understood that the use of certain of theforegoing excipients, carriers, or stabilizers will result in theformation of VEGF-2 salts.

VEGF-2 to be used for therapeutic administration must be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeutic VEGF-2compositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

VEGF-2 ordinarily will be stored in unit or multi-dose containers, forexample, sealed ampules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10 ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous VEGF-2 solution, and the resultingmixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized VEGF-2 using bacteriostaticWater-for-Injection.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods fortreating disorders, diseases and conditions. The gene therapy methodsrelate to the introduction of nucleic acid (DNA, RNA and antisense DNAor RNA) sequences into an animal to achieve expression of the VEGF-2polypeptide of the present invention. This method requires apolynucleotide which codes for a VEGF-2 polypeptide operatively linkedto a promoter and any other genetic elements necessary for theexpression of the polypeptide by the target tissue. Such gene therapyand delivery techniques are known in the art, see, for example,WO90/11092, WO98/11779; U.S. Pat. Nos. 5,693,622, 5,705,151, 5,580,859;Tabata H. et al. (1997) Cardiovasc. Res. 35(3):470-479, Chao, J et al.(1997) Pharmacol. Res. 35(6):517-522, Wolff, J. A. (1997) Neuromuscul.Disord. 7(5):314-318, Schwartz, B. et al. (1996) Gene Ther.3(5):405-411, Tsurumi, Y. et al. (1996) Circulation 94(12):3281-3290(incorporated herein by reference).

As discussed more fully below, the VEGF-2 polynucleotide sequencespreferably have a therapeutic effect after being taken up by a cell.Examples of polynucleotides that are themselves therapeutic areanti-sense DNA and RNA; DNA coding for an anti-sense RNA; or DNA codingfor tRNA or rRNA to replace defective or deficient endogenous molecules.For example, a promoter may be operably linked to a DNA sequenceencoding for an antisense RNA. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of an mRNAmolecule into a polypeptide (Okano, J. Neurochem 56:560 (1991)). Theantisense RNA must be of sufficient length and complementarity toprevent translation of its target mRNA.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to aVEGF-2 polynucleotide ex vivo, with the engineered cells then beingprovided to a patient to be treated with the polypeptide. Such methodsare well-known in the art. For example, see Belldegrun, A., et al., J.Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., CancerResearch 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato,L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al.,Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer GeneTherapy 3: 31-38 (1996)), which are herein incorporated by reference. Inone embodiment, the cells which are engineered are photoreceptor cells.These engineered cells may be reintroduced into the patient throughdirect injection to the tissue of origin, the tissues surrounding thetissue of origin, veins or arteries, or through catheter injection. Inone embodiment, the engineered cells are attached to the sclera toproduce and release VEGF-2 protein directly into the vitreous humor.

Photoreceptor cell transplantation studies designed to replace defectiveor lost cells due to retinal disease or damage have been performedsuccessfully in animal models of retinal degeneration (Silverman andHughes, Invest. Ophthalmol. Vis. Sci. 30:1684-1690(1989); Gouras et al.,Neuro-Ophthalmol. 10:165-176 (1990)). It is contemplated thatphotoreceptor cells may be obtained from donor eyes and maintained inculture as described herein. The cells would then be used as a source ofpurified photoreceptors to be transplanted via the subretinal space intothe retina of patients suffering from retinal disease or damage. Thesepatients will be treated with immunosuppressive therapies to eliminateimmunological responses and rejection of the grafted cells. The ex vivodonor retinas will be cultured in the presence of VEGF-2, in order toenhance their growth and survival. The patients that will receivephotoreceptor cell transplants will be treated with intravitreal VEGF-2needed to promote the survival and the maturation of the graftedphotoreceptors.

As discussed in more detail below, the VEGF-2 polynucleotide constructscan be delivered by any method that delivers injectable materials to thecells of an animal, such as, injection into the interstitial space oftissues (heart, muscle, skin, lung, liver, and the like). The VEGF-2polynucleotide constructs may be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

In one embodiment, the VEGF-2 polynucleotide is delivered as a nakedpolynucleotide. The term “naked” polynucleotide, DNA or RNA refers tosequences that are free from any delivery vehicle that acts to assist,promote or facilitate entry into the cell, including viral sequences,viral particles, liposome formulations, lipofectin™ or precipitatingagents and the like. However, the VEGF-2 polynucleotides can also bedelivered in liposome formulations and lipofectin™ formulations and thelike can be prepared by methods well known to those skilled in the art.Such methods are described, for example, in U.S. Pat. Nos. 5,593,972,5,589,466, and 5,580,859, which are herein incorporated by reference. U.S. Pat. No. 5,770,580 describes gene therapy methods for delivery intothe eye.

The VEGF-2 polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication.Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSGavailable from Stratagene; pSVK3, pBPV, pMSG and pSVL available fromPharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of VEGF-2 DNA. Suitable promoters includeadenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs; the b-actin promoter; and human growthhormone promoters. The promoter also may be the native promoter forVEGF-2.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The VEGF-2 polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Especially preferred is the eye. Interstitial space of thetissues comprises the intercellular, fluid, mucopolysaccharide matrixamong the reticular fibers of organ tissues, elastic fibers in the wallsof vessels or chambers, collagen fibers of fibrous tissues, or that samematrix within connective tissue ensheathing muscle cells or in thelacunae of bone. It is similarly the space occupied by the plasma of thecirculation and the lymph fluid of the lymphatic channels. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked acid sequence injection, an effective dosage amount of DNAor RNA will be in the range of from about 0.05 mg/kg body weight toabout 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues, especially the eye.However, other parenteral routes may also be used, such as, inhalationof an aerosol formulation particularly for delivery to lungs orbronchial tissues, throat or mucous membranes of the nose. In addition,naked VEGF-2 DNA constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure. The nakedpolynucleotides are delivered by also be delivered by topicaladministration and so-called “gene guns”. These delivery methods areknown in the art.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin™,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the VEGF-2 polynucleotide constructs arecomplexed in a liposome preparation. Liposomal preparations for use inthe instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA(1987) 84:7413-7416, which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which isherein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is hereinincorporated by reference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin™,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated byreference). Other commercially available liposomes include transfectace(DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication No. WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P. Felgneret al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15 EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art. The liposomes can comprisemultilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), orlarge unilamellar vesicles (LUVs), with SUVs being preferred. Thevarious liposome-nucleic acid complexes are prepared using methods wellknown in the art. See, e.g., Straubinger et al., Methods of Immunology(1983), 101:512-527, which is herein incorporated by reference. Forexample, MLVs containing nucleic acid can be prepared by depositing athin film of phospholipid on the walls of a glass tube and subsequentlyhydrating with a solution of the material to be encapsulated. SUVs areprepared by extended sonication of MLVs to produce a homogeneouspopulation of unilamellar liposomes. The material to be entrapped isadded to a suspension of preformed MLVs and then sonicated.

When using liposomes containing cationic lipids, the dried lipid film isresuspended in an appropriate solution such as sterile water or anisotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and thenthe preformed liposomes are mixed directly with the DNA. The liposomeand DNA form a very stable complex due to binding of the positivelycharged liposomes to the cationic DNA. SUVs find use with small nucleicacid fragments. LUVs are prepared by a number of methods, well known inthe art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilsonet al., Cell (1979) 17:77); ether injection (Deamer, D. and Bangham, A.,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); detergent dialysis (Enoch, H. and Strittmatter, P.,Proc. Natl. Acad. Sci. USA (1979) 76:145); and reverse-phase evaporation(REV) (Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka, F. andPapahadjopoulos, D., Proc. Natl. Acad. Sci. USA (1978) 75:145;Schaefer-Ridder et al., Science (1982) 215:166), which are hereinincorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ration will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication no. WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication no. WO 94/9469 (which areherein incorporated by reference) provide methods for deliveringDNA-cationic lipid complexes to mammals.

In certain embodiments, cells are be engineered, ex vivo or in vivo,using a retroviral particle containing RNA which comprises a sequenceencoding VEGF-2. Retroviruses from which the retroviral plasmid vectorsmay be derived include, but are not limited to, Moloney Murine LeukemiaVirus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus,avian leukosis virus, gibbon ape leukemia virus, human immunodeficiencyvirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, φ-2,φ-AM, PA12, T19-14X, VT-19-17-H2, φCRE, φCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990),which is incorporated herein by reference in its entirety. The vectormay transduce the packaging cells through any means known in the art.Such means include, but are not limited to, electroporation, the use ofliposomes, and CaPO₄ precipitation. In one alternative, the retroviralplasmid vector may be encapsulated into a liposome, or coupled to alipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding VEGF-2. Such retroviral vectorparticles then may be employed, to transduce eukaryotic cells, either invitro or in vivo. The transduced eukaryotic cells will express VEGF-2.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with VEGF-2 polynucleotide contained in an adenovirus vector. Adenoviruscan be manipulated such that it encodes and expresses VEGF-2, and at thesame time is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. Adenovirus expression is achieved withoutintegration of the viral DNA into the host cell chromosome, therebyalleviating concerns about insertional mutagenesis. Furthermore,adenoviruses have been used as live enteric vaccines for many years withan excellent safety profile (Schwartz, A. R. et al. (1974) Am. Rev.Respir. Dis. 109:233-238). Finally, adenovirus mediated gene transferhas been demonstrated in a number of instances including transfer ofalpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M.A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell68:143-155). Furthermore, extensive studies to attempt to establishadenovirus as a causative agent in human cancer were uniformly negative(Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992);Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al.,Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress the VEGF-2 polynucleotide of interest which is operably linkedto a promoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol.158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The VEGF-2 polynucleotideconstruct is inserted into the AAV vector using standard cloningmethods, such as those found in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAVvector is then transfected into packaging cells which are infected witha helper virus, using any standard technique, including lipofection,electroporation, calcium phosphate precipitation, etc. Appropriatehelper viruses include adenoviruses, cytomegaloviruses, vacciniaviruses, or herpes viruses. Once the packaging cells are transfected andinfected, they will produce infectious AAV viral particles which containthe VEGF-2 polynucleotide construct. These viral particles are then usedto transduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the VEGF-2 polynucleotide construct integrated intoits genome, and will express VEGF-2.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding VEGF-2) via homologous recombination (see, e.g., U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No.WO 96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989).This method involves the activation of a gene which is present in thetarget cells, but which is not normally expressed in the cells, or isexpressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the VEGF-2 desired endogenous polynucleotide sequenceso the promoter will be operably linked to the endogenous sequence uponhomologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous VEGF-2 sequence is placedunder the control of the promoter. The promoter then drives theexpression of the endogenous VEGF-2 sequence.

Preferably, the polynucleotide encoding VEGF-2 contains a secretorysignal sequence that facilitates secretion of the protein. Typically,the signal sequence is positioned in the coding region of thepolynucleotide to be expressed towards or at the 5′ end of the codingregion. The signal sequence may be homologous or heterologous to thepolynucleotide of interest and may be homologous or heterologous to thecells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a protein-coated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers (Kaneda et al.,Science 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian.

Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals and birds. Preferred mammals includehumans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs,with humans being particularly preferred.

Nucleic Acid Utilities

VEGF-2 nucleic acid sequences and VEGF-2 polypeptides may also beemployed for in vitro purposes related to scientific research, synthesisof DNA and manufacture of DNA vectors, and for the production ofdiagnostics and therapeutics to treat human disease. For example, VEGF-2may be employed for in vitro culturing of photoreceptor cells, where itis added to the conditional medium in a concentration from 10 pg/ml to10 ng/ml.

Fragments of the full length VEGF-2 gene may be used as a hybridizationprobe for a CDNA library to isolate other genes which have a highsequence similarity to the gene or similar biological activity. Probesof this type generally have at least 50 base pairs, although they mayhave a greater number of bases. The probe may also be used to identify acDNA clone corresponding to a full length transcript and a genomic cloneor clones that contain the complete VEGF-2 gene including regulatory andpromoter regions, exons, and introns. An example of a screen comprisesisolating the coding region of the VEGF-2 gene by using the known DNAsequence to synthesize an oligonucleotide probe. Labeledoligonucleotides having a sequence complementary to that of the gene ofthe present invention are used to screen a library of human cDNA,genomic DNA or MRNA to determine which members of the library the probehybridizes to.

This invention provides methods for identification of VEGF-2 receptors.The gene encoding the receptor can be identified by numerous methodsknown to those of skill in the art, for example, ligand panning and FACSsorting (Coligan et al., Current Protocols in Immun., 1(2), Chapter 5,(1991)). Preferably, expression cloning is employed whereinpolyadenylated RNA is prepared from a cell responsive to VEGF-2, and acDNA library created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to VEGF-2.Transfected cells which are grown on glass slides are exposed to labeledVEGF-2. VEGF-2 can be labeled by a variety of means including iodinationor inclusion of a recognition site for a site-specific protein kinase.Following fixation and incubation, the slides are subjected toautoradiographic analysis. Positive pools are identified and sub-poolsare prepared and retransfected using an iterative sub-pooling andrescreening process, eventually yielding a single clone that encodes theputative receptor.

As an alternative approach for receptor identification, labeled VEGF-2can be photoaffinity linked with cell membrane or extract preparationsthat express the receptor molecule. Cross-linked material is resolved byPAGE and exposed to X-ray film. The labeled complex containing VEGF-2 isthen excised, resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the gene encoding the putativereceptor.

Examples

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples, certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 mg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 Fl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 mgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37 EC are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.8:4057 (1980).

“Oligonucleotides” refer to either a single stranded polydeoxynucleotideor two complementary polydeoxynucleotide strands, which may bechemically synthesized. Such synthetic oligonucleotides have no 5′phosphate and thus will not ligate to another oligonucleotide withoutadding a phosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989), p. 146). Unless otherwiseprovided, ligation may be accomplished using known buffers andconditions with 10 units of T4 DNA ligase (“ligase”) per 0.5 mg ofapproximately equimolar amounts of the DNA fragments to be ligated.

Unless otherwise stated, transformation was performed as described bythe method of Graham, F. and Van der Eb, A., Virology 52:456-457 (1973).

Example 1 Expression Pattern of VEGF-2 in Human Tissues and BreastCancer Cell Lines

Northern blot analysis was carried out to examine the levels ofexpression of VEGF-2 in human tissues and breast cancer cell lines inhuman tissues. Total cellular RNA samples were isolated with RNAzol™ Bsystem (Biotecx Laboratories, Inc.). About 10 mg of total RNA isolatedfrom each breast tissue and cell line specified was separated on 1%agarose gel and blotted onto a nylon filter, (Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)). The labeling reaction wasdone according to the Stratagene Prime-It kit with 50 ng DNA fragment.The labeled DNA was purified with a Select-G-50 column from 5 Prime÷3Prime, Inc (Boulder, Colo.). The filter was then hybridized with aradioactive labeled full length VEGF-2 gene at 1,000,000 cpm/ml in 0.5 MNaPO₄ and 7% SDS overnight at 65° C. After washing twice at roomtemperature and twice at 60° C. with 0.5× SSC, 0.1% SDS, the filterswere then exposed at −70° C. overnight with an intensifying screen. Amessage of 1.6 Kd was observed in 2 breast cancer cell lines. FIG. 5,lane #4 represents a very tumorigenic cell line that is estrogenindependent for growth.

Also, 10 mg of total RNA from 10 human adult tissues were separated onan agarose gel and blotted onto a nylon filter. The filter was thenhybridized with radioactively labeled VEGF-2 probe in 7% SDS, 0.5 MNaPO4, pH 7.2; 1% BSA overnight at 65° C. Following washing in 0.2× SSCat 65° C., the filter was exposed to film for 24 days at −70° C. withintensifying screen. See FIG. 6.

Example 2 Expression of the Truncated Form of VEGF-2 (SEQ ID NO:4) by InVitro Transcription and Translation

The VEGF-2 cDNA was transcribed and translated in vitro to determine thesize of the translatable polypeptide encoded by the truncated form ofVEGF-2 and a partial VEGF-2 cDNA. The two inserts of VEGF-2 in thepBluescript SK vector were amplified by PCR with three pairs ofprimers, 1) M13-reverse and forward primers; 2) M13-reverse primer andVEGF primer F4; and 3) M13-reverse primer and VEGF primer F5. Thesequence of these primers are as follows.

M13-2 reverse primer: 5′-ATGCTTCCGGCTCGTATG-3′ (SEQ ID NO: 11)

This sequence is located upstream of the 5′ end of the VEGF-2 cDNAinsert in the pBluescript vector and is in an anti-sense orientation asthe cDNA.

A T3 promoter sequence is located between this primer and the VEGF-2cDNA.

M13-2 forward primer: 5′GGGTTTTCCCAGTCACGAC-3′ (SEQ ID NO: 12)

This sequence is located downstream of the 3′ end of the VEGF-2 cDNAinsert in the pBluescript vector and is in an anti-sense orientation asthe cDNA insert.

VEGF primer F4: 5′-CCACATGGTTCAGGAAAGACA-3′ (SEQ ID NO: 13)

This sequence is located within the VEGF-2 cDNA in an anti-senseorientation from bp 1259-1239, which is about 169 bp away from the 3′end of the stop codon and about 266 bp before the last nucleotide of thecDNA.

PCR reaction with all three pairs of primers produce amplified productswith T3 promoter sequence in front of the cDNA insert. The first andthird pairs of primers produce PCR products that encode the polypeptideof VEGF-2 shown in SEQ ID NO:4. The second pair of primers produce PCRproduct that misses 36 amino acids coding sequence at the C-terminus ofthe VEGF-2 polypeptide.

Approximately 0.5 mg of PCR product from first pair of primers, 1 mgfrom second pair of primers, 1 mg from third pair of primers were usedfor in vitro transcription/translation. The in vitrotranscription/translation reaction was performed in a 25 Fl of volume,using the T_(N)TJ Coupled Reticulocyte Lysate Systems (Promega, CAT#L4950). Specifically, the reaction contains 12.5 Fl of T_(N)T rabbitreticulocyte lysate 2 Fl of T_(N)T reaction buffer, 1 Fl of T3polymerase, 1 Fl of 1 mM amino acid mixture (minus methionine), 4 Fl of³⁵S-methionine (>1000 Ci/mmol, 10 mCi/ml), 1 Fl of 40 U/μl; RNasinribonuclease inhibitor, 0.5 or 1 mg of PCR products. Nuclease-free H₂Owas added to bring the volume to 25 Fl. The reaction was incubated at30° C. for 2 hours. Five microliters of the reaction product wasanalyzed on a 4-20% gradient SDS-PAGE gel. After fixing in 25%isopropanol and 10% acetic acid, the gel was dried and exposed to anX-ray film overnight at 70° C.

As shown in FIG. 7, PCR products containing the truncated VEGF-2 cDNA(i.e., as depicted in SEQ ID NO:3) and the cDNA missing 266 bp in the 3′un-translated region (3′-UTR) produced the same length of translatedproducts, whose molecular weights are estimated to be 38-40 kd (lanes 1and 3). The cDNA missing all the 3′UTR and missing sequence encoding theC-terminal 36 amino acids was translated into a polypeptide with anestimated molecular weight of 36-38 kd (lane 2).

Example 3 Cloning and Expression of VEGF-2 Using the BaculovirusExpression System

The DNA sequence encoding the VEGF-2 protein without 46 amino acids atthe N-terminus, see ATCC™ No. 97149, was amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene:

The 5′ primer has the sequence TGT AAT ACG ACT CAC TAT AGG GAT CCC GCCATG GAG GCC ACG GCT TAT GC (SEQ ID NO:14) and contains a BamH1restriction enzyme site (in bold) and 17 nucleotide sequencecomplementary to the 5′ sequence of VEGF-2 (nt. 150-166).

The 3′ primer has the sequence GATC TCT AGA TTA GCT CAT TTG TGG TCT (SEQID NO:15) and contains the cleavage site for the restriction enzyme XbaIand 18 nucleotides complementary to the 3′ sequence of VEGF-2, includingthe stop codon and 15 nt sequence before stop codon.

The amplified sequences were isolated from a 1% agarose gel using acommercially available kit (“Geneclean™,” BIO 101, Inc., La Jolla,Calif.). The fragment was then digested with the endonuclease BamH1 andXbaI and then purified again on a 1% agarose gel. This fragment wasligated to pAcGP67A baculovirus transfer vector (Pharmingen) at theBamH1 and XbaI sites. Through this ligation, VEGF-2 cDNA was cloned inframe with the signal sequence of baculovirus gp67 gene and was locatedat the 3′ end of the signal sequence in the vector. This is designatedpAcGP67A-VEGF-2.

To clone VEGF-2 with the signal sequence of gp67 gene to the pRG1 vectorfor expression, VEGF-2 with the signal sequence and some upstreamsequence were excised from the pAcGP67A-VEGF-2 plasmid at the Xhorestriction endonuclease site located upstream of the VEGF-2 cDNA and atthe XbaI restriction endonuclease site by XhoI and XbaI restrictionenzyme. This fragment was separated from the rest of vector on a 1%agarose gel and was purified using “Geneclean™” kit. It was designatedF2.

The PRG1 vector (modification of pVL941 vector) is used for theexpression of the VEGF-2 protein using the baculovirus expression system(for review see: Summers, M. D. and Smith, G. E., “A Manual of Methodsfor Baculovirus Vectors and Insect Cell Culture Procedures,” TexasAgricultural Experimental Station Bulletin No. 1555, (1987)). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases BamH1, Sma1,XbaI, Bg1II and Asp718. A site for restriction endonuclease Xho1 islocated upstream of BamH1 site. The sequence between Xho1 and BamH1 isthe same as that in PAcGp67A (static on tape) vector. Thepolyadenylation site of the simian virus (SV)40 is used for efficientpolyadenylation. For an easy selection of recombinant virus thebeta-galactosidase gene from E. coli is inserted in the same orientationas the polyhedrin promoter followed by the polyadenylation signal of thepolyhedrin gene. The polyhedrin sequences are flanked at both sides byviral sequences for the cell-mediated homologous recombination ofcotransfected wild-type viral DNA. Many other baculovirus vectors couldbe used in place of pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers, M. D., Virology 170:31-39 (1989).

The plasmid was digested with the restriction enzymes XboI and XbaI andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The DNA was then isolated from a 1% agarose gel usingthe commercially available kit (“Geneclean™” BIO 101 Inc., La Jolla,Calif.). This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNAligase. E. coli HB101 cells were then transformed and bacteriaidentified that contained the plasmid (pBac gp67-VEGF-2) with the VEGF-2gene using the enzymes BamH1 and XbaI. The sequence of the clonedfragment was confirmed by DNA sequencing.

5 mg of the plasmid pBac gp67-VEGF-2 was cotransfected with 1.0 mg of acommercially available linearized baculovirus (“BaculoGold™J baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofectin™ method(Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)).

1 mg of BaculoGold™J virus DNA and 5 mg of the plasmid pBac gp67-VEGF-2were mixed in a sterile well of a microtiter plate containing 50 ml ofserum free Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 ml Lipofectin™ plus 90 ml Grace's medium were added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture was added dropwise to the Sf9 insect cells (ATCC™ CRL 1711)seeded in a 35 mM tissue culture plate with 1 ml Grace's medium withoutserum. The plate was rocked back and forth to mix the newly addedsolution. The plate was then incubated for 5 hours at 27° C. After 5hours the transfection solution was removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum was added.The plate was put back into an incubator and cultivation continued at27° C. for four days.

After four days the supernatant was collected and a plaque assayperformed similar as described by Summers and Smith, supra. As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10). Fourdays after the serial dilution, the virus was added to the cells, bluestained plaques were picked with the tip of an Eppendorf pipette. Theagar containing the recombinant viruses was then resuspended in anEppendorf tube containing 200 ml of Grace's medium. The agar was removedby a brief centrifugation and the supernatant containing the recombinantbaculovirus was used to infect Sf9 cells seeded in 35 mM dishes. Fourdays later the supernatants of these culture dishes were harvested andthen stored at 4° C.

Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-gp67-VEGF-2 at a multiplicity of infection (MOI) of 1. Sixhours later the medium was removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 mCi of ³⁵S-methionine and 5 mCi ³⁵S cysteine (Amersham)were added. The cells were further incubated for 16 hours before theywere harvested by centrifugation and the labeled proteins visualized bySDS-PAGE and autoradiography.

Protein from the medium and cytoplasm of the Sf9 cells was analyzed bySDS-PAGE under non-reducing and reducing conditions. See FIGS. 8A and8B, respectively. The medium was dialyzed against 50 mM MES, pH 5.8.Precipitates were obtained after dialysis and resuspended in 100 mM NaCitrate, pH 5.0. The resuspended precipitate was analyzed again bySDS-PAGE and was stained with Coomassie Brilliant Blue. See FIG. 9.

The medium supernatant was also diluted 1:10 in 50 mM MES, pH 5.8 andapplied to an SP-650M column (1.0×6.6 cm, Toyopearl) at a flow rate of 1ml/min. Protein was eluted with step gradients at 200, 300 and 500 mMNaCl. The VEGF-2 was obtained using the elution at 500 mM. The eluatewas analyzed by SDS-PAGE in the presence or absence of reducing agent,b-mercaptoethanol and stained by Coomassie Brilliant Blue. See FIG. 10.

Example 4 Expression of Recombinant VEGF-2 in COS Cells

The expression of plasmid, VEGF-2-HA is derived from a vector pcDNAI/Amp(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gene, 3) E. coli replication origin, 4) CMV promoter followedby a polylinker region, an SV40 intron and polyadenylation site. A DNAfragment encoding the entire VEGF-2 precursor and a HA tag fused inframe to its 3′ end was cloned into the polylinker region of the vector,therefore, the recombinant protein expression is directed under the CMVpromoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein as previously described (Wilson et al.,Cell 37:767 (1984)). The infusion of HA tag to the target protein allowseasy detection of the recombinant protein with an antibody thatrecognizes the HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding VEGF-2, ATCC™ No. 97149, was constructed byPCR using two primers: the 5′ primer (CGC GGA TCC ATG ACT GTA CTC TACCCA) (SEQ ID NO:16) contains a BamH1 site followed by 18 nucleotides ofVEGF-2 coding sequence starting from the initiation codon; the 3′sequence (CGC TCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA CTC GAGGCT CAT TTG TGG TCT 3′) (SEQ ID NO:17) contains complementary sequencesto an XbaI site, HA tag, XhoI site, and the last 15 nucleotides of theVEGF-2 coding sequence (not including the stop codon). Therefore, thePCR product contains a BamHI site, coding sequence followed by an XhoIrestriction endonuclease site and HA tag fused in frame, a translationtermination stop codon next to the HA tag, and an XbaI site. The PCRamplified DNA fragment and the vector, pcDNAI/Amp, were digested withBamH1 and XbaI restriction enzyme and ligated. The ligation mixture wastransformed into E. coli strain SURE (Stratagene Cloning Systems, LaJolla, Calif. 92037) the transformed culture was plated on ampicillinmedia plates and resistant colonies were selected. Plasmid DNA wasisolated from transformants and examined by restriction analysis for thepresence of the correct fragment. For expression of the recombinantVEGF-2, COS cells were transfected with the expression vector byDEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Theexpression of the VEGF-2-HA protein was detected by radiolabelling andimmunoprecipitation method (E. Harlow and D. Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cellswere labeled for 8 hours with ³⁵S-cysteine two days post transfection.Culture media was then collected and cells were lysed with detergent(RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mMTris, pH 7.5) (Wilson et al., Cell 37:767 (1984)). Both cell lysate andculture media were precipitated with an HA specific monoclonal antibody.Proteins precipitated were analyzed on 15% SDS-PAGE gels.

Example 5 Construction of Amino Terminal and Carboxy Terminal DeletionMutants

In order to identify and analyze biologically active VEGF-2polypeptides, a panel of deletion mutants of VEGF-2 was constructedusing the expression vector pHE4a.

1. Construction of VEGF-2 T103-L215 in pHE4

To permit Polymerase Chain Reaction directed amplification andsub-cloning of VEGF-2 T103-L215 (amino acids 103 to 215 in FIG. 1 or SEQID NO:2) into the E. coli protein expression vector, pHE4, twooligonucleotide primers complementary to the desired region of VEGF-2were synthesized with the following base sequence:

5′ Primer (Nde I/START and 18 nt of coding sequence):

(SEQ ID NO: 18) 5′-GCA GCA CAT ATG ACA GAA GAG ACT ATA AAA-3′

3′ Primer (Asp718, STOP, and 15 nt of coding sequence):

(SEQ ID NO: 19) 5′-GCA GCA GGT ACC TCA CAG TTT AGA CAT GCA-3′

The above described 5′ primer (SEQ ID NO:18), incorporates an NdeIrestriction site and the above described 3′ Primer (SEQ ID NO:19),incorporates an Asp718 restriction site. The 5′ primer (SEQ ID NO:18)also contains an ATG sequence adjacent and in frame with the VEGF-2coding region to allow translation of the cloned fragment in E. coli,while the 3′ primer (SEQ ID NO:19) contains one stop codon(preferentially utilized in E. coli) adjacent and in frame with theVEGF-2 coding region which ensures correct translational termination inE. coli.

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature VEGF-2 (aa 24-419 in SEQ ID NO:2) as, for example,constructed in Example 3 as template. The resulting amplicon wasrestriction digested with NdeI and Asp718 and subcloned into NdeI/Asp718digested pHE4a expression vector.

2. Construction of VEGF-2 T103-R227 in pHE4

To permit Polymerase Chain Reaction directed amplification andsub-cloning of VEGF-2 T103-R227 (amino acids 103 to 227 in FIG. 1 or SEQID NO:2) into the E. coli protein expression vector, pHE4, twooligonucleotide primers complementary to the desired region of VEGF-2were synthesized with the following base sequence:

5′ Primer (Nde I/START and 18 nt of coding sequence):

(SEQ ID NO: 20) 5′-GCA GCA CAT ATG ACA GAA GAG ACT ATA AAA-3′

3′ Primer (Asp 718, STOP, and 15 nt of coding sequence):

(SEQ ID NO: 21) 5′-GCA GCA GGT ACC TCA ACG TCT AAT AAT GGA-3′

In the case of the above described primers, an NdeI or Asp718restriction site was incorporated he 5′ primer and 3′ primer,respectively. The 5′ primer (SEQ ID NO:20) also contains an ATG sequenceadjacent and in frame with the VEGF-2 coding region to allow translationof the cloned fragment in E. coli, while the 3′ Primer (SEQ ID NO:21)contains one stop codon (preferentially utilized in E. coli) adjacentand in frame with the VEGF-2 coding region which ensures correcttranslational termination in E. coli.

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature VEGF-2 (aa 24-419 in SEQ ID NO:2) as, for example,constructed in Example 3, as template. The resulting amplicon wasrestriction digested with NdeI and Asp718 and subcloned into NdeI/Asp718digested pHE4a protein expression vector.

3. Construction of VEGF-2 T103-L215 in pA2GP

In this illustrative example, the plasmid shuttle vector pA2 GP is usedto insert the cloned DNA encoding the N-terminal and C-terminal deletedVEGF-2 protein (amino acids 103-215 in FIG. 1 or SEQ ID NO:2), into abaculovirus to express the N-terminal and C-terminal deleted VEGF-2protein, using a baculovirus leader and standard methods as described inSummers et al., A Manual of Methods for Baculovirus Vectors and InsectCell Culture Procedures, Texas Agricultural Experimental StationBulletin No. 1555 (1987). This expression vector contains the strongpolyhedrin promoter of the Autographa californica nuclear polyhedrosisvirus (AcMNPV) followed by the secretory signal peptide (leader) of thebaculovirus gp67 protein and convenient restriction sites such as BamHI,Xba I and Asp718. The polyadenylation site of the simian virus 40(“SV40”) is used for efficient polyadenylation. For easy selection ofrecombinant virus, the plasmid contains the beta-galactosidase gene fromE. coli under control of a weak Drosophila promoter in the sameorientation, followed by the polyadenylation signal of the polyhedringene. The inserted genes are flanked on both sides by viral sequencesfor cell-mediated homologous recombination with wild-type viral DNA togenerate viable virus that expresses the cloned polynucleotide.

Many other baculovirus vectors could be used in place of the vectorabove, such as pAc373, pVL941 and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39 (1989).

The cDNA sequence encoding the VEGF-2 protein without 102 amino acids atthe N-terminus and without 204 amino acids at the C-terminus in FIG. 1,was amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ sequences of the gene.

The 5′ primer has the sequence 5′-GCA GCA GGA TCC CAC AGA AGA GAC TATAAA-3′ (SEQ ID NO:22) containing the BamHI restriction enzyme site (inbold) followed by 1 spacer nt to stay in-frame with the vector-suppliedsignal peptide, and 17 nt of coding sequence bases of VEGF-2 protein.The 3′ primer has the sequence 5′-GCA GCA TCT AGA TCA CAG TTT AGA CATGCA-3′ (SEQ ID NO:23) containing the XbaI restriction site (in bold)followed by a stop codon and 17 nucleotides complementary to the 3′coding sequence of VEGF-2.

The amplified sequences were isolated from a 1% agarose gel using acommercially available kit (“Geneclean™,” BIO 101, Inc., La Jolla,Calif.). The fragment was then digested with the endonuclease BamH1 andXbaI and then purified again on a 1% agarose gel. This fragment wasligated to pA2 GP baculovirus transfer vector (Supplier) at the BamH1and XbaI sites. Through this ligation, VEGF-2 cDNA representing theN-terminal and C-terminal deleted VEGF-2 protein (amino acids 103-215 inFIG. 1 or SEQ ID NO:2) was cloned in frame with the signal sequence ofbaculovirus GP gene and was located at the 3′ end of the signal sequencein the vector. This is designated pA2GPVEGF-2.T103-L215.

4. Construction of VEGF-2 T103-R227 in pA2GP

The cDNA sequence encoding the VEGF-2 protein without 102 amino acids atthe N-terminus and without 192 amino acids at the C-terminus in FIG. 1(i.e., amino acids 103-227 of SEQ ID NO:2) was amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene.

The 5′-GCA GCA GGA TCC CAC AGA AGA GAC TAT AAA ATT TGC TGC-3′ primer hasthe sequence (SEQ ID NO:24) containing the BaniHI restriction enzymesite (in bold) followed by 1 spacer nt to stay in-frame with thevector-supplied signal peptide, and 26 nt of coding sequence bases ofVEGF-2 protein. The 3′ primer has the sequence 5′-GCA GCA TCT AGA TCAACG TCT AAT AAT GGA ATG AAC-3′ (SEQ ID NO:25) containing the XbaIrestriction site (in bold) followed by a stop codon and 21 nucleotidescomplementary to the 3′ coding sequence of VEGF-2.

The amplified sequences were isolated from a 1% agarose gel using acommercially available kit (“Geneclean™,” BIO 101, Inc., La Jolla,Calif.). The fragment was then digested with the endonuclease BamH1 andXbaI and then purified again on a 1% agarose gel. This fragment wasligated to pA2 GP baculovirus transfer vector (Supplier) at the BamH1and XbaI sites. Through this ligation, VEGF-2 cDNA representing theN-terminal and C-terminal deleted VEGF-2 protein (amino acids 103-227 inFIG. 1 or SEQ ID NO:2) was cloned in frame with the signal sequence ofbaculovirus GP gene and was located at the 3′ end of the signal sequencein the vector. This construct is designated pA2GPVEGF-2.T103-R227.

5. Construction of VEGF-2 in pC1

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology,438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart etal., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with therestriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate thecloning of the gene of interest. The vectors contain in addition the 3Nintron, the polyadenylation and termination signal of the ratpreproinsulin gene.

The vector pC1 is used for the expression of VEGF-2 protein. Plasmid pC1is a derivative of the plasmid pSV2-dhfr [ATCC™ Accession No. 37146].Both plasmids contain the mouse DHFR gene under control of the SV40early promoter. Chinese hamster ovary—or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Life Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J Biol. Chem.253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097:107-143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology9:64-68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene it is usually co-amplified and over-expressed. It is stateof the art to develop cell lines carrying more than 1,000 copies of thegenes. Subsequently, when the methotrexate is withdrawn, cell linescontain the amplified gene integrated into the chromosome(s).

Plasmid pC1 contains for the expression of the gene of interest a strongpromoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus(Cullen, et al., Molecular and Cellular Biology, March 1985:438-4470)plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530, 1985).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: BamHI, Pvu11,and Nru1. Behind these cloning sites the plasmid contains translationalstop codons in all three reading frames followed by the 3N intron andthe polyadenylation site of the rat preproinsulin gene. Other highefficient promoters can also be used for the expression, e.g., the humanb-actin promoter, the SV40 early or late promoters or the long terminalrepeats from other retroviruses, e.g., HIV and HTLVI. For thepolyadenylation of the mRNA other signals, e.g., from the human growthhormone or globin genes can be used as well.

Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC1 is digested with the restriction enzyme BamHI and thendephosphorylated using calf intestinal phosphates by procedures known inthe art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding VEGF-2, ATCC™ Accession No. 97149, wasconstructed by PCR using two primers corresponding to the 5′ and 3′ endsof the VEGF-2 gene: the 5′ Primer (5′-GAT CGA TCC ATC ATG CAC TCG CTGGGC TTC TTC TCT GTG GCG TGT TCT CTG CTC G-3′ (SEQ ID NO:26)) contains aKlenow-filled BamHI site and 40 nt of VEGF-2 coding sequence startingfrom the initiation codon; the 3′ primer (5′-GCA GGG TAC GGA TCC TAG ATTAGC TCA TTT GTG GTC TTT-3′ (SEQ ID NO:27)) contains a BamHI site and 16nt of VEGF-2 coding sequence not including the stop codon.

The PCR amplified DNA fragment is isolated from a 1% agarose gel asdescribed above and then digested with the endonuclease BaniHI and thenpurified again on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 cells are then transformed and bacteria identified that containedthe plasmid pC1. The sequence and orientation of the inserted gene isconfirmed by DNA sequencing. This construct is designated pC1VEGF-2.

6. Construction of pC4SigVEGF-2 T103-L215

Plasmid pC4Sig is plasmid pC4 (Accession No. 209646) containing a humanIgG Fc portion as well as a protein signal sequence.

To permit Polymerase Chain Reaction directed amplification andsub-cloning of VEGF-2 T103-L215 (amino acids 103 to 215 in FIG. 1 or SEQID NO:2) into pC4Sig, two oligonucleotide primers complementary to thedesired region of VEGF-2 were synthesized with the following basesequence:

5′ Primer (Bam HI and 26 nt of coding sequence):

(SEQ ID NO: 28) 5′-GCA GCA GGA TCC ACA GAA GAG ACT ATA AAA TTT GCT GC-3′

3′ Primer (Xba I, STOP, and 15 nt of coding sequence):

(SEQ ID NO: 29) 5′-CGT CGT TCT AGA TCA CAG TTT AGA CAT GCA TCG GCA G-3′

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature VEGF-2 (aa 24-419) as, for example, constructed in Example 3,as template. The resulting amplicon was restriction digested with BaniHIand XbaI and subcloned into BamHI/XbaI digested pC4Sig vector.

7. Construction of pC4SigVEGF-2 T103-R227

To permit Polymerase Chain Reaction directed amplification andsub-cloning of VEGF-2 T103-L215 (amino acids 103 to 227 in FIG. 1 or SEQID NO:2) into pC4Sig, two oligonucleotide primers complementary to thedesired region of VEGF-2 were synthesized with the following basesequence:

5′ Primer (Bam HI and 26 nt of coding sequence):

(SEQ ID NO: 30) 5′-GCA GCA GGA TCC ACA GAA GAG ACT ATA AAA TTT GCT GC-3′

3′ Primer (Xba I, STOP, and 21 nt of coding sequence):

(SEQ ID NO: 31) 5′-GCA GCA TCT AGA TCA ACG TCT AAT AAT GGA ATG AAC-3′

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature VEGF-2 (aa 24-419) as, for example, constructed in Example 3,as template. The resulting amplicon was restriction digested with BamHIand XbaI and subcloned into BamHI/XbaI digested pC4Sig vector.

8. Construction of pC4VEGF-2 M1-M263

The expression vector pC4 contains the strong promoter (LTR) of the RousSarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447(March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamiHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vector contains in addition the 3N intron, thepolyadenylation and termination signal of the rat preproinsulin gene.

In this illustrative example, the cloned DNA encoding the C-terminaldeleted VEGF-2 M1-M263 protein (amino acids 1-263 in FIG. 1 or SEQ IDNO:2) is inserted into the plasmid vector pC4 to express the C-terminaldeleted VEGF-2 protein.

To permit Polymerase Chain Reaction directed amplification andsub-cloning of VEGF-2 M1-M263 into the expression vector, pC4, twooligonucleotide primers complementary to the desired region of VEGF-2were synthesized with the following base sequence:

5′ Primer (SEQ ID NO: 32) 5′-GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTTCTT CTC-3′ 3′ Primer (SEQ ID NO: 33) 5′-GAC TGG TAC CTT ATC ACA TAA AATCTT CCT GAG CC-3′

In the case of the above described 5′ primer, an BamH1 restriction sitewas incorporated, while in the case of the 3′ primer, an Asp718restriction site was incorporated. The 5′ primer also contains 6 nt, 20nt of VEGF-2 coding sequence, and an ATG sequence adjacent and in framewith the VEGF-2 coding region to allow translation of the clonedfragment in E. coli, while the 3′ primer contains 2 nt, 20 nt of VEGF-2coding sequence, and one stop codon (preferentially utilized in E. coli)adjacent and in frame with the VEGF-2 coding region which ensurescorrect translational termination in E. coli.

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature VEGF-2 (aa 24-419) as constructed, for example, in Example 3as template. The resulting amplicon was restriction digested with BamH1and Asp718 and subcloned into BamH1/Asp718 digested pC4 proteinexpression vector. This construct is designated pC4VEGF-2 M1-M263.

9. Construction of pC4VEGF-2 M1-D311

In this illustrative example, the cloned DNA encoding the C-terminaldeleted VEGF-2 Ml -D311 protein (amino acids 1-311 in FIG. 1 or SEQ IDNO:2) is inserted into the plasmid vector pC4 to express the C-terminaldeleted VEGF-2 protein.

To permit Polymerase Chain Reaction directed amplification andsub-cloning of VEGF-2 M1-D311 into the expression vector, pC4, twooligonucleotide primers complementary to the desired region of VEGF-2were synthesized with the following base sequence:

5′ Primer (SEQ ID NO: 34) 5′-GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTTCTT CTC-3′ 3′ Primer (SEQ ID NO: 35) 5′-GAC TGG TAC CTT ATC AGT CTA GTTCTT TGT GGG G-3′

In the case of the above described 5′ primer, an BamH1 restriction sitewas incorporated, while in the case of the 3′ primer, an Asp718restriction site was incorporated. The 5′ primer also contains 6 nt, 20nt of VEGF-2 coding sequence, and an ATG sequence adjacent and in framewith the VEGF-2 coding region to allow translation of the clonedfragment in E. coli, while the 3′ primer contains 2 nt, 20 nt of VEGF-2coding sequence, and one stop codon (preferentially utilized in E. coli)adjacent and in frame with the VEGF-2 coding region which ensurescorrect translational termination in E. coli.

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature VEGF-2 (aa 24-419) as constructed, for example, in Example 3as template. The resulting amplicon was restriction digested with BamH1and Asp718 and subcloned into BamH1/Asp718 digested pC4 proteinexpression vector.

10. Construction of pC4VEGF-2 M1-Q367

In this illustrative example, the cloned DNA encoding the C-terminaldeleted VEGF-2 M1-D311 protein (amino acids 1-311 in SEQ ID NO:2) isinserted into the plasmid vector pC4 to express the C-terminal deletedVEGF-2 protein.

To permit Polymerase Chain Reaction directed amplification andsub-cloning of VEGF-2 M1-D311 into the expression vector, pC4, twooligonucleotide primers complementary to the desired region of VEGF-2were synthesized with the following base sequence:

5′ Primer (SEQ ID NO: 36) 5′-GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTTCTT CTC-3′ 3′ Primer (SEQ ID NO: 37) 5′-GAC TGG TAC CTC ATT ACT GTG GACTTT CTG TAC ATT C-3′

In the case of the above described 5′ primer, an BamH1 restriction sitewas incorporated, while in the case of the 3′ primer, an Asp718restriction site was incorporated. The 5′ primer also contains 6 nt, 20nt of VEGF-2 coding sequence, and an ATG sequence adjacent and in framewith the VEGF-2 coding region to allow translation of the clonedfragment in E. coli, while the 3′ primer contains 2 nt, 20 nt of VEGF-2coding sequence, and one stop codon (preferentially utilized in E. coli)adjacent and in frame with the VEGF-2 coding region which ensurescorrect translational termination in E. coli.

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature VEGF-2 (aa 24-419) as constructed, for example, in Example 3as template. The resulting amplicon was restriction digested with BamH1and Asp718 and subcloned into BamH1/Asp718 digested pC4 proteinexpression vector. This construct is designated pC4VEGF-2 M1-Q367.

Example 6 Method of Treatment Using Gene Therapy for Production ofVEGF-2 Polypeptide—In Vivo

Suitable template DNA for production of mRNA coding for VEGF-2 isprepared in accordance with a standard recombinant DNA methodology.Sterile and endotoxin-free oligonucleotides are diluted in Sterile andendotoxin-free oligonucleotides are diluted in Balanced Salt Solution(BSS, Alcon, Fort Worth, Tex.) so as to have the same pH and electrolyteconcentration as the aqueous or vitreous of the eye. Emalphor EC620(2.5%, GAF Corp.) (Bursell et al. (1993) J. Clin. Invest. 92:2872-2876),a petroleum product, is added to change viscosity and aid in deliveryproperties. Doses to achieve intravitreal concentrations ranging from0.1 μM-100 μM are administered. The volume delivered is between 1 μl and1 ml depending on the volume of the eye.

The intubated patient is Anesthetized with fluorane. The face and eyesare prepared with a betadine scrub and draped in the usual sterilefashion. The sterile polynucleotide with vehicle is injected with a 33gauge needle on a sterile syringe at the posterior limbus (pars plana)through full thickness sclera into the vitreous. No closing suture isrequired unless there is leakage. Antibiotic drops containing gentamicinor erythromycin ointment is applied to the surface of the globe in thepalpebral fissure several times per day until there is complete woundclosure. The frequency of injection ranges from every other day to onceevery 6 months or less, depending on the severity of the diseaseprocess, the degree of intraocular inflammation, the character of thevehicle (i.e., slow release characteristics), and the tolerance of theeye to injections. Short and long term follow-up check-ups for possibleretinal detachment from the injections are necessary.

The eye upon dilation is monitored for signs of inflammation, infection,and photoreceptor growth by both an direct and a indirect ophthalmoscopeto view the retina and fundus. Monitoring can be as frequent as everyday in cases where premature infants are threatened with retinaldetachment. The frequency of monitoring will diminish with resolution ofdisease.

The patient is treated weekly with intraocular injections ofpolynucleotide resuspended in the appropriate vehicle (BSS, Emanfour) atconcentrations within the range of 0.1 to 100 μM. This treatment may besupplemented with systemic delivery of polynucleotide (i.e.,intravenous, subcutaneous, or intramuscular) from 2 to 5 times per dayto once a month.

Example 7 Method of Treatment Using Gene Therapy—Ex vivo HomologousRecombination

Photoreceptor cells are obtained from a subject by biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. To construct aplasmid for targeting to the VEGF-2 locus, plasmid pUC 18 (MBIFermentas, Amherst, N.Y.) is digested with HindIII. The CMV promoter isamplified by PCR with an XbaI site on the 5′ end and a BamHI site on the3′ end. Two VEGF-2 non-coding sequences are amplified via PCR: oneVEGF-2 non-coding sequence (VEGF-2 fragment 1) is amplified with aHindIII site at the 5′ end and an Xba site at the 3′ end; the otherVEGF-2 non-coding sequence (VEGF-2 fragment 2) is amplified with a BamHIsite at the 5′ end and a HindIII site at the 3′ end. The CMV promoterand VEGF-2 fragments are digested with the appropriate enzymes (CMVpromoter-XbaI and BamHI; VEGF-2 fragment 1-XbaI; VEGF-2 fragment2-BamHI) and ligated together. The resulting ligation product isdigested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5.×10⁶ cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and250-300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 EC. The following day, the media is aspiratedand replaced with 10 ml of fresh media and incubated for a further 16-24hours.

The engineered photoreceptor cells are then injected into the host. Thephotoreceptor cells now produce the protein product.

Example 8 VEGF-2 Activity on Retinal Cells

The retina has proven to be an advantageous experimental model forstudying the role of intrinsic and extrinsic factors in the regulationof the development of neuronal and non-neuronal cell types from a moreprimitive neuroepithelial cell. The differentiated retina is composed ofseven cell types: sensory (rod and cone photoreceptors), glia (Mütllercells), and two types of neurons, interneurons (horizontal, bipolar, andamacrine), and projection neurons (ganglion cells) (for review seeDowling, 1987). The development of the various cell types in the retinadoes not occur synchronously with the majority of the cones, andganglion and horizontal cells developing before birth (for review seeAltshuler et al., 1991; Harris, 1991; Reh, 1991). In contrast,differentiation of a majority of the rods, the main cell type in the ratretina, occurs postnatally. Clonal analysis of the progeny of retinalprecursor cells has demonstrated that the progenitor cells can producevarious combinations of retinal cell types indicating that theprogenitors are either totipotent or multipotent depending on thedevelopmental age examined (Turner and Cepko, 1987; Turner et al., 1990;Wetts and Fraser, 1998). Furthermore, findings from both in vivo and invitro studies demonstrate that the final phenotype of the retinal cellsis largely lineage independent which suggest that the changingmicroenvironment within the retina has a role in determining thecellular potential of the progenitor cells as well as the differentiatedphenotype of the progeny (Watanabe and Raff, 1990, 1992; Harris, 1991;Reh, 1991; Ezzeddine et al. 1997).

In vitro, retinal cell proliferation and differentiation is regulated bya variety of factors; for example, FGF-2 (Hicks and Courtois, 1992),CNTF (Ezzeddine et al., 1997; Fuhrmann et al., 1995), LIF (Ezzeddine etal., 1997), TGF_(Lilleen and Cepko, 1992), retinoic acid (Kelly et al.,1994), and EGF (Lillien, 1995). Recently, Yang and Cepko (1996) haveidentified and characterized the expression pattern of VEGFR-2/FLK-1 indeveloping and adult retina. VEGFR transcripts are first detected atE11.5 in association with the developing retinal vasculature and withthe central region of the neural retina. By developmental day E15,VEGFR-2 expression extends to the periphery of the retina consistentwith the outward gradient of retinal development (Young, 1985; LaVail etal., 1991). VEGFR-2 expression was largely localized to the ventricularzone during the perinatal period when neurogenesis is at its peak and alarge number of post-mitotic neurons are being formed.

As shown below, the major in vitro effect of VEGF is during earlydevelopment and involves the proliferation of multipotent progenitorcells since the level of BrdU and the number of photoreceptor andamacrine cells are increased. VEGF-2 enhanced the proliferation ofretinal cells derived from E15 embryos and the magnitude of the responseincreased with age. The early proliferative response to VEGF-2administration was not effected by CNTF. However, CNTF did inhibit theVEGF-2 induced increase in the level of rhodopsin protein.

Experimental Procedures

Animals. Timed pregnant animals are obtained from Harlan Sprague-Dawley(Indianapolis, Ind.). All animal related procedures are conducted instrict compliance with approved institutional protocols and inaccordance with provisions for animal care and use described in theGuide for the Care and Use of Laboratory Animals (NIH publication No.86-23,1985).

Retinal Cultures The retinal tissue are obtained from either lateembryonic or neonatal rats. The dissociated primary cells are preparedby incubating the tissue in 0.25% trypsin for 6 min at 37° C. Followingthe inactivation of the trypsin by a 5 min incubation in growth medium(F12:Dulbecco's modified Eagle's medium (DMEM) containing 1% fetalbovine serum, 1% hormonal supplements (N2, Bottenstein, 1983), 1%glutamine and 0.5% penicillin-streptomycin (10,000 units/ml and 10mg/ml, respectively, Gibco, Grand Island, N.Y.) containing 50 μg/mldeoxyribonuclease type I (Sigma, St. Louis, Mo.), the tissue fragmentsare passed repeatedly through a Pasteur pipette with a constricted tipof a diameter of approximately 1 mm. The dissociated cells are collectedby centrifugation (800× g, 5 min) and resuspended in growth medium. Thecells are seeded in 96 well plates precoated with poly-L-lysine (50μg/ml, Sigma) and laminin (10 μg/ml, Gibco), at a density of 425cells/mm² unless stated otherwise. The cultures are gradually shifted togrowth medium without serum by changing one-half of the medium everyother day. The trophic factors are replenished with each medium change.

Hippocampal Astrocytes Purified cultures of astrocytes are prepared fromrat hippocampi using a method previously described (Greene et al.,1998).

Rhodopsin Immunohistochemistry and Cell Counting Procedures For theimmunohistochemical staining, the cultures are fixed overnight in 4%paraformaldehyde containing 4% sucrose. For rhodopsin and syntaxinstaining, the cultures are permeabilized with 0.05% saponin in PBS for30 min. The non-specific IgG binding is inhibited by incubating thecells in PBS containing 5% horse serum and 2% BSA for 3 h at roomtemperature. The cultures are then incubated overnight at 4° C. withanti-rhodopsin (1:10,000, Rho 4D2, Dr. Molday, University of BritishColumbia) or anti-syntaxin (1:10,000, Sigma) diluted in PBS containing5% horse serum and 2% BSA (Molday, 1989). Following the removal of theprimary antibody, the cultures are incubated with a biotinylatedanti-mouse antibody (1:2,500) for 90 min. The avidin-biotin-peroxidasecomplex, diluted 1:50 in PBS containing 5% horse serum and 2% BSA, isthen added for 60 minutes. To visualize the bound peroxidase,diaminobenzidine is used at a final concentration of 0.4 mg/ml in a 0.1Macetate buffer containing 2.5% nickel sulfate. The number ofimmunopositive cells per well is determined by counting the labeledcells in an area representing 11% of the total surface area of the welland then corrected for the total surface area.

BrdU Immunohistochemistry The retinal cells are incubated with BrdU for4 h and subsequently, washed twice with PBS. Growth medium is then addedback to the cultures, which are then maintained in vitro for varioustime intervals. At the end of the incubation period, the cultures arefixed and immunohistochemically stained for the incorporated BrdU as permanufacturer's (Boehringer Mannheim, Indianapolis, Ind.) instructions.

Rhodopsin and GFAP ELISAs For the rhodopsin ELISA, the cultures arerinsed with PBS and fixed overnight with 4% paraformaldehyde containing4% sucrose. The cultures are then rinsed with PBS and permeabilized with0.05% saponin in PBS for 30 min at room temperature. The non-specificprotein binding sites are suppressed by incubating the cells in PBScontaining 5% horse serum and 2% BSA (blocking buffer) for at least 3 h.The cultures are incubated overnight with the mouse anti-rhodopsinantibody (diluted 1:500 in blocking buffer) and then subsequently, withgoat anti-mouse IgG conjugated to horseradish peroxidase (diluted1:2,500) in blocking solution. The cultures are washed extensively withPBS and then the substrate (3,3′, 5,5′-tetramethylbenzidine) is added tothe wells and the plates were incubated in the dark for 60 min. Thereaction is stopped by adding 2 M H₂SO₄ and the amount of product formedis quantitated by measuring the absorbance at 450 nm. The absorption ofthe reagent blank ranged from 0.1 to 0.15 and is not subtracted from theindicated values. The GFAP ELISA is conducted essentially as previouslydescribed (Greene et al., 1998).

Cell survival assay based on Calcein AM measurements. At the end of theincubation period the cultures were rinsed once with Ham's F-12. Thecalcein AM was added to a final concentration of 2 μM in 100 μl of Ham'sF-12 and the cultures were incubated for 60 min at 37 degree C. At theend of the incubation period the cultures were rinsed. The absorbance at530 mn was determined on an ELISA plate reader.

High-affinity GABA uptake The level of high-affinity GABA uptake isdetermined as previously described (Greene et al., 1998).

[³H]Thymidine Incorporation The cultures are treated with trophicfactors for 24 h and during the last 4 h, the cells are labeled with[³H]thyrnidine, at a final concentration of 0.33 μM (25 Ci/mmol,Amersham, Arlington Heights, Ill.). The incorporated [³H]thymidine isprecipitated with ice-cold 10% trichloroacetic acid for 24 h.Subsequently, the cells are rinsed with ice-cold water. Following lysisin 0.5 M NaOH, the lysates and PBS rinses (500 μl) are pooled, andcounted.

Results

The regulatory role of VEGFs on photoreceptor cell development isinitially investigated using cultures derived from postnatal day 1 (PN1)animals. Previous reports have demonstrated that multipotent progenitorsare present during this developmental period and retain their capacityto differentiate into photoreceptor cells as well as other retinal celltypes in vitro (Marrow et al., 1998). Treatment with VEGF-2 or VEGF-1 (Rand D Systems, Minneapolis, Minn.) induces a dose- and time-dependentincrease in the level of rhodopsin protein in the retinal cultures (FIG.14A). The time course of the VEGF-induced increase in rhodopsin isrelatively slow, consistent with the known developmental profile ofphotoreceptor cells. After 5 days of treatment, a 25-40% increase inrhodopsin protein is noted with 10 to 100 ng/ml of VEGF-2. However, by 7to 9 days of treatment, these same concentrations of VEGF-2 produced a200-250% increase in rhodopsin protein. Furthermore, at these later timepoints, concentrations of VEGF as low as 1 ng/ml significantly increasedrhodopsin levels. Changes in the amount of rhodopsin may reflect changesin the level of expression of the protein or changes in the number ofphotoreceptor cells or both. To ascertain if VEGF treatment effected thetotal number of retinal cells, the level of emission of calcein AM ismonitored. An approximate 25-50% increase in calcein emission isobserved after a 5-7 day treatment with at least 10 ng/ml of VEGF-2(FIG. 14B). A pronounced increase in the basal level of calcein emissionis noted in the retinal cultures between 7 and 9 days indicating thatretinal cell proliferation that is independent of exogenous VEGF hadincreased by this later time point. The presence of VEGF-2, even atconcentrations as high as 100 ng/ml, for 9 days did not further increasethe number of retinal cells. However, there is not a concomitantincrease in the basal level of rhodopsin protein after 9 days in culturesuggesting that the proliferation of other cell types accounts for theincrease in the level of calcein emission (FIG. 14A). Treatment withVEGF-1 induced similar changes in the level of rhodopsin and calceinemission to those described for VEGF-2 (FIGS. 14C and 14D,respectively).

To determine if the increase in rhodopsin content and calcein emissionreflected an increase in the number of photoreceptor cells, cultures aretreated with VEGF-1 or VEGF-2 for 9 days and then immunohistochemicallystained for rhodopsin. To quantitate the effects of VEGF treatment, cellcounts are made. The number of rhodopsin immunopositive cells increasedas a function of concentration with the response having an EC50 value of0.25 and 1.5 ng/ml for VEGF-1 and VEGF-2, respectively (FIG. 15). At asaturating concentration of VEGF-2, a 2.4-fold increase in the number ofrods is observed. Furthermore, the response is stable in the presence ofconcentrations of VEGF-2 as high as 100 ng/ml suggesting that VEGF-2does not readily induce a desensitization of the biological response.The dose response observed with VEGF-1 is similar to that obtained withVEGF-2 which is consistent with the results from the rhodopsin ELISAs.

The mechanism by which VEGF-2 induces an increase in photoreceptor cellnumber may involve an increase in the proliferation of precursor cells,enhanced survival of differentiated photoreceptor cells, and/or theredirection of the rod lineage pathway. To investigate if VEGF-2 ismitogenic for retinal cells, the cultures are treated with factors 4 hafter plating and then subsequently labeled with BrdU after 24, 48 or 72h. At the end of the final labeling period (72 h), the cultures arefixed and the incorporated BrdU is immunohistochemically detected. Asignificant increase in the number of BrdU labeled cells is not observeuntil after 48 h of treatment, when 10 ng/ml of VEGF-2 or VEGF-1 induceda 2- to 3-fold increase (FIGS. 16A and 16B, respectively). The EC50value for the response is calculated as 1 ng/ml. The 48 h time pointappeared near maximal since after an incubation of 72 h, the level ofBrdU incorporation had declined in the VEGF treated cultures regardlessof concentration. However, this decline in the number of immunopositivecells is not specifically related to VEGF administration. The basallevel of BrdU incorporation decreased from 1300 to 700 immunopositivecells per well suggesting that a general loss in the proliferativeactivity of the retinal progenitor cells is occurring during this timeperiod. In spite of the lower over-all proliferative activity in thecultures at the later time points, VEGF administration still resulted ina 2-fold increase in the number of BrdU labeled cells. Similar resultsare obtained using [³H]thymidine incorporation (FIG. 16C).

To further characterize a role for VEGF during the early in vitroculture period, the effect that delaying the addition of the factor tothe cultures had on the level of rhodopsin protein is examined. Theinitial addition of VEGF is made 4, 24, or 48 h after plating and thecultures are subsequently maintained for 9 days and then prepared forthe rhodopsin ELISA. The loss of the response to VEGF-2 or VEGF-1 as afunction of the time lapsed between the isolation of the cells and theinitial addition of the factors is depicted in FIGS. 17A and 17B,respectively. The addition of factors within 4 h of the plating of thecells resulted in a 3-fold increase in the level of rhodopsin. However,delaying the initial treatment with VEGF by 24 or 48 h resulted in thereduction of the maximal response by 28 and 43%, respectively.Furthermore, after a delay of 48 h, only treatment with 100 ng/ml ofVEGF-2 induced a significant increase in rhodopsin content. Thussuggesting that the proliferative effect that the VEGFs are having onretinal cells is developmentally restricted and involves theproliferation of photoreceptor progenitors.

The possibility that VEGF effects other retinal cell types that are bornpostnatally e.g. amacrine and Müller cells, is also investigated. Themorphology of amacrine cells, identified on the basis of theirexpression of syntaxin, is examined. (Data not shown). Treatment witheither VEGF induced a dose-dependent increase in the number of syntaxinimmunopositive cells with 10 ng/ml inducing a maximal increase ofapproximately 2.4-fold as compared to the vehicle treated controls (FIG.18A). In contrast to the results with the rhodopsin ELISA and cellcounts, 100 ng/ml of VEGF-1 or -2 induced a smaller increase in thenumber of syntaxin immunopositive cells than is observed with 10 ng/ml.To further characterize the effect VEGF-2 treatment on the phenotype ofamacrine cells, the level of high-affinity GABA uptake is measured. FIG.18B depicts the dose-dependent increase in GABA uptake induced by VEGF.The dose response curve is similar to that observed when using thenumber of immunopositive syntaxin cells as the endpoint with asignificant increase and saturation in GABA uptake occurring with 1 and10 ng/ml of VEGF-2, respectively.

Müller glial cells are identified on the basis of their expression ofglial fibillary acidic protein (Björklund et al., 1985). To determine ifVEGF-2 has an effect on the number of Müller cells or on the level ofdifferentiation, the amount of GFAP protein is measured by ELISA. After7 days in culture, there is no significance difference in the level ofGFAP, when comparing treatment with factors versus the vehicle control(FIG. 18C). Furthermore, treatment with VEGF-2 did not increase thenumber of endothelial cells, immunopositive cells, in the retinalcultures (data not shown).

To further characterize the developmental pattern of the VEGF response,retinal cells are isolated at different developmental stages, and themitogenic response to VEGF-2 is quantitated after 48 h by labeling thecultures with [³H]thymidine. In addition, as it has been notedpreviously that the differentiation of photoreceptor cells in vitro isdensity dependent, the effect that plating density has on the responseto VEGF is also investigated.

When the cultures are derived from E15 animals and plated at a densityof 212 cells/mm², the basal level of [³H]thymidine incorporation is1589±94 dpm/well and treatment with VEGF-2 induced a maximal increase of50% (FIG. 19A). In contrast to the dose response observed with P1cultures where saturation occurs at 10 ng/ml, the proliferative responsein the E15 cultures saturates at a concentration of 1 ng/ml.Furthermore, there is an inverse relationship between the platingdensity and the mitogenic response to VEGF-2. At a density of 318cells/mm², a leftward shift in the dose response curve is noted withconcentrations higher than 1 ng/ml causing a desensitization of theresponse. At the highest density tested (425 cells/mm²), the retinalcells are unresponsive to VEGF-2. It is interesting to note that FGF-2(10 ng/ml), which has a similar biological activity as VEGF-2 in the P1cultures (see below), inhibited the proliferation in the E15 cultures byas much as 62% in the higher density cultures (data not shown).

In cultures derived from E20 animals the basal level of [³H]thymidineincorporation at a plating density of 212 cells/mm² is 3361±192 and thelevel of stimulation of [³H]thymidine incorporation with VEGF-2treatment is generally greater, ranging up to 80-100%, at the lowerplating densities (FIG. 19B). There is still a trend toward VEGF-2having less of an effect in cultures plated at the highest density.However, the inhibitory effect is much less pronounced. By P1, where thebasal level of [³H]thymidine incorporation is 478±33, there is aleftward shift in the dose response with saturation occurring at 10ng/ml and the extent of the maximal increase is greater, in the range of300% (FIG. 19C). Furthermore, there is no discernible effect of platingdensity on the response to VEGF-2.

To characterize more fully the responsiveness of the rod or rodprogenitor cells, the effect that EGF, FGF-2 or TGFβ-1 has on the numberof retinal cells and on the level of rhodopsin protein is compared tothat achieved with VEGF-2. EGF, a mitogen for various cell types,induces a 31% increase in the number of retinal cells with the responsesaturating at 1 ng/ml and remaining stable up to 100 ng/ml (FIG. 20A).However, there is no concomitant increase in the level of rhodopsinprotein in the EGF treated cultures (FIG. 20B). FGF-2 in a concentrationrange of 1-100 ng/ml induces a small increase (13%) in the number ofretinal cells. Furthermore, FGF-2, which activates a number of the FGFreceptors, induces an increase in the level of rhodopsin protein. A 45%increase in the level of rhodopsin is observed with concentrations ofFGF-2 as low as 1 ng/ml resulting in an EC50 value for the response inthe range of 0.5 ng/ml. Treatment with TGFβ-1 results in a decrease inboth the number of retinal cells and the level of rhodopsin protein. Ata concentration of 0.1 ng/ml, TGFβ-1 maximally decreased calceinexpression and the level of rhodopsin protein by 40 and 90%,respectively.

The results from the BrdU labeling experiments demonstrate that VEGF-2enhances the rate of proliferation of retinal progenitor cells. Sincethe developmental pathway of photoreceptor cells is thought to belineage independent and thus under the regulation of environmentalfactors (Ezzeddine ZD et al., 1997), VEGF may also modulatephotoreceptor cell development at additional downstream sites. It hasbeen determined previously that CNTF inhibits the differentiation ofphotoreceptor cells relatively late in their developmental pathway byredirecting their phenotype toward the bipolar cell lineage. Toinvestigate the potential interaction of the two factors by co-treatingretinal cultures with VEGF-2 at a concentration that is saturating forthe induction of photoreceptor cells and various concentrations of CNTF.The increase in rhodopsin protein induced by VEGF-2 is inhibited by CNTFin a dose-dependent manner (FIG. 21A). The inhibitory response had anIC50 value of 0.4 ng/ml and treatment with 100 ng/ml of CNTF resulted inthe complete inhibition of the VEGF-2 response. However, treatment withCNTF did not alter the total number of retinal cells in the cultures(FIG. 21B). To determine if the inhibitory effect of CNTF is an early orlate event, the effect that co-administration of CNTF had on theincreased level of [³H]thymidine incorporation induced by VEGF-2 istested. In contrast to the previous results, the addition of CNTF didnot inhibit the VEGF induced proliferative response (FIG. 21C). Thesefindings further substantiate that these two factors regulatephotoreceptor cell development at different points in the lineagepathway.

Discussion

The above experiments identify and characterize the effect of VEGF-1 andVEGF-2 on retinal cells in vitro. Treatment with VEGF in thesub-nanomolar range induces an increase in the number of photoreceptorand amacrine cells as well as increases the level of rhodopsin proteinand high-affinity GABA uptake. Time course studies demonstrate that VEGFinduces a maximal increase in [³H]thymidine incorporation within 48 h ofits addition and delaying the treatment of the cultures by 24-48 hresults in the loss of the proliferative and differentiation responses.The mitogenic response was developmentally regulated with VEGF-2inducing an increase in [³H]thymidine incorporation with cells derivedfrom E15, E20 and P1 animals. In comparison with members of othertrophic factor families, the response to treatment with VEGF-2 and FGF-2were similar in that both factors increased the level of rhodopsinprotein without inducing an increase in the total number of cells after9 days in culture. The co-administration of CNTF with VEGF-2 resulted inthe inhibition of the VEGF induced increase in the level of rhodopsinbut not in the proliferative response.

The VEGF receptor family is currently composed of four members (forreview see Klagsbrun and D'Amore, 1996; Wen et al., 1998). The receptorsdemonstrate distinct yet overlapping ligand specificity. VEGFR-1 (Flt-1)and VEGFR-2 (Flk-1) bind the various forms of VEGF-1; while, VEGFR-2 andVEGFR-3 (Flt-4) bind VEGF-2. Thus both VEGF-1 and VEGF-2 activateVEGFR-2 (Joukov et al., 1998) and both ligands have similar biologicalactivities in the retinal cultures. Recently, Yang and Cepko (1996)described the developmental expression pattern of VEGFR-2 in the retina.Extent from the expected expression of the receptor on the newly formingvasculature, receptors were also present on components of the neuralretina. This expression pattern is maintained during development as theretina grows in a centripetal manner.

The effect of developmental age on the response of the retinalprogenitor cells to VEGF is consistent with the developmental expressionpattern of the receptor (Yang and Cepko, 1996). Mitogenic effects ofVEGF, based on [³H]thymidine incorporation studies, were noted at theearliest developmental time point examined, E15, as well as at E20 andP1. The magnitude of the proliferative effect increased with agereaching a peak by P1. VEGF-2 is more efficacious on E15 and E20cultures than at P1 since that response saturated at 1 as opposed to 10ng/ml, respectively. Furthermore, the basal level of proliferation invitro also changed with developmental age with the highest levelsobserved at E17. The finding that the basal level of proliferation wasrelatively low at E15 but increased 4-fold with a 2-fold increase incell density, a greater proportional increase than was observed at theother developmental ages, suggests that endogenous mitogens may underliethe desensitization that occurs with VEGF-2 treatment in the E15cultures. Moreover, these data indicate that increased levels of VEGFduring early development may have a negative impact on thedifferentiation of photoreceptor cells. The influence of developmentalage on the response of retinal progenitor cells to other growth factorshas also been observed (Altshuler and Cepko, 1992). Lillien and Cepko(1992) reported that the proliferative response of retinal cells inmonolayer cultures to FGF-1 and FGF-2 was greater at earlier gestationalages (e.g. E15 and E18) and by E21 or P0 a rightward shift in the doseresponse curve was apparent.

Previous studies in goldfish and frog have suggested that amacrine celldevelopment is regulated by cell-cell contact (Negishi et al., 1982; Rehand Tully, 1986). More recently, the importance of cell-cell contact forthe in vitro development of photoreceptor cells was also described byWantabe and Raff (1990, 1992) in reaggregated cultures and then later byAltshuler and Cepko (1992) with dissociated retinal cells plated incollagen gels. In the former study, when E15 retinal cells werereaggregated with a 50-fold excess of neonatal retinal cells, there wasno change in the developmental time when the rhodopsin immunopositivecells were observed. However, there was a significant increase in theproportion of the E15 cells that eventually differentiated intophotoreceptor cells. In the case of the monolayer cultures used in thisstudy, there is a dissociation between the VEGF-2 induced earlyproliferative response and the later differentiation of photoreceptorcells. For example, VEGF-2 increases [³H]thymidine incorporation by3-4-fold in cultures seeded at densities as low as 212 cells/mm² andtreatment for 7 days resulted in cell densities equivalent to thoseachieved at the higher plating densities (e.g. 425 cells/mm²). However,there was no detectable rhodopsin protein or immunopositive cells inthese cultures. These results suggest that there is not only a criticalcell-cell interaction necessary for the development of photoreceptorcells but also a time frame during which the stimulus produced via cellcontact is probable necessary.

Comparing the time course of the VEGF-induced proliferation to thedevelopmental time course of the appearance of rhodopsin proteinindicates that there is an approximate 5 day lag between the two events.The appearance of rhodopsin protein likely reflects the induction ofgene transcription since the two events have been shown to be closelycorrelated (Treisman et al., 1988). This time interval is similar tothat observed by Morrow et al. (1998) in vivo and in vitro studies whenconsidering progenitor cells derived from animals within an age range ofE20 to P3. Furthermore between 5 and 9 days in vitro, we observed thegreatest increase in the level of rhodopsin protein and this time periodis within the postnatal developmental period (day 6-10) in vivo duringwhich there is a pronounced appearance of rhodopsin immunopositive cells(Morrow et al., 1998). The correlation in these developmental timewindows suggests that although VEGF-2 induces the proliferation ofphotoreceptor progenitor cells, it does not induce a significant delayin the differentiation of photoreceptor cells. As might be expected ifthe progenitor cells were prevented from leaving the cell cycle.

In comparison to members of other trophic factor families, the responseto VEGF-2 resembled that of FGF-2 in that both factors increased thelevel of rhodopsin protein while inducing relatively small increases inthe total number of retinal cells after 9 days in vitro. In addition, aproliferative response, based on [³H]thymidine incorporation and cellcounts, to FGF-2 was noted by Lillien and Cepko (1992) as late as P3suggesting that FGF-2 retains some mitogenic activity in postnatalcultures. In contrast to our findings with VEGF-2, Fontaine et al.(1998) demonstrated that FGF-2 also has a survival effect onphotoreceptor cells derived from P5 animals (data not shown). TGFβ-1treatment resulted in a decrease in both the number of retinal cells andthe level of rhodopsin protein. Kimichi et al. (1988) reported similarobservations using human fetal retinal cultures with the exception thatmaximal inhibition with the human cells required 0.5 ng/ml of TGFB-1 ascompared to the less than 0.1 ng/ml required in the rodent cultures.

CNTF, a member of the neuropoietic family of cytokines, is known toeffect the development of photoreceptor cells in vitro and in vivo andto enhance the survival of photoreceptor cells following light-induceddamage (Unoki and LaVail, 1994; Fuhrman et al., 1995; Ezzeddine et al.,1997; Cayouette et al., 1998). In contrast to CNTF, VEGF-2 did notrescue photoreceptor cells in the constant light-induced damage model(LaVail et al., 1992; Wen et al., 1995; R. Wen and R. Alderson,unpublished data). Treatment of postnatal rat retinal explant cultureswith CNTF results in an increase in the number of cells expressingbipolar cell markers with a loss in the population of cells expressingrhodopsin. Analysis of the effect of CNTF on the fate of [³H]thymidinelabeled P0 retinal cells suggests that the cytokine does not induce theproliferation or increase the survival of this cell population(Ezzeddine et al., 1997). Furthermore, the initiation of the effect ofCNTF occurred at about the time that the cells became post-mitotic andbegin to express rhodopsin. These data are consistent with the findingsreported here demonstrating that CNTF inhibits the VEGF-2 inducedincrease in rhodopsin protein observed between 5 and 7 days in culture,but not its mitogenic activity observed between 1 and 2 days.

During the course of development in the retina, oxygen levels controlthe microarchitecture of retinal vessels that in turn match the patternof differentiation of retinal neurons (Chan-Ling et al., 1990; Phelps,1990). Stone et al. (1995) have demonstrated that in the retina,astrocytes and microglia respond to hypoxia by synthesizing andsecreting VEGF which in turn induces vessel formation. The studiesreported here suggest that the early differentiation events regulated byVEGF involve not only vessel formation but also photoreceptor progenitorcell proliferation. This ultimately may result in the coordinateddevelopment of numerous cell types in the retina.

Example 9 Enhanced Response of Endothelial Cells to VEGF-2 and AntibodyCo-Treatment

Antibodies generated by HGS have been shown to bind to VEGF-2 by ELISAassays, but are not thought to bind to the sites involved in receptorinteractions. Monoclonal 13D was mapped to an epitope on the N-terminalside of the molecule and monoclonal 13A2 was mapped to an epitope on theC-terminal end. (See FIG. 24). The polyclonal antibody recognizes anumber of different sites but it is not believed to bind to the segmentof the active protein which interacts with the receptor. Theseantibodies were used to quantitatively determine stimulation orinhibition of the proliferation of bovine lymphatic endothelial cells(LEC) by co-treating with VEGF-2 and the above antibodies.

An alamar blue™ LEC proliferation assay was used, which incorporates afluorometric growth indicator based on detection of metabolic activity.The Alamar blue™ is an oxidation-reduction indicator that bothfluoresces and changes color in response to chemical reduction of growthmedium resulting from cell growth. As cells grow in culture, innatemetabolic activity results in a chemical reduction of the immediatesurrounding environment. Reduction related to growth causes theindicator to change from oxidized (non-fluorescent blue) form to reduced(fluorescent red) form. i.e. stimulated proliferation will produce astronger signal and inhibited proliferation will produce a weaker signaland the total signal is proportional to the total number of cells.

The materials used for the alamar blue™ LEC proliferation assay include:Alamar Blue™ (Biosource Cat #DAL1100); DMEM 10%FBS+PennStrep+Glutamine+75 mg BBE+45 mg Heparin (Growth media); DMEM 10%FBS+PennStrep+Glutamine (Starvation Media); DMEM 0.5%FBS+PennStrep+Glutamine (sample dilution media); CytoFluor™ Fluorescencereader; 96 well plate(s); and LEC cells.

The alamar blue™ assay was performed as follows. For timing purposes itis best to seed the cells onto the 96 well plate(s) on a Wednesday,change to starvation media on Thursday, inoculate samples on Friday,incubate over the weekend then add alamar blue™ incubate and read onMonday.

LEC cells were seeded in growth media at a density of 5000 cells/well ofa 96 well plate and placed at 37° C. overnight. After the overnightincubation of the LEC cells, the growth media was removed and replacedwith starvation medium and incubated for another 24 hours at 37° C.After the second 24 hour incubation, the cells were inoculated with theappropriate dilutions of protein sample(s) (prepared in DMEM+0.5% FBS)in triplicate wells. Once the cells have been inoculated with thesamples the plate was placed back in the 37° C. incubator for threedays.

After three days, 10 μl of stock alamar blue™ was added to each well andthe plate was placed back in the 37° C. incubator for four hours. Theplate was then read at 530 nm excitation and 590 nm emission using theCytoFluor™ fluorescence reader. Direct output is recorded in relativefluorescence units.

The background level of activity was observed with the starvation mediumalone. This is compared to the output observed from the positive controlsamples (VEGF-1 and/or bFGF) and the HGS protein dilutions.

Three different antibody preparations made by HGS (2 mouse monoclonals,13A2, 13D6 and a rabbit polyclonal antibody) were evaluated for theirability to modulate the response of LECs to VEGF-2 mediated activation.It was previously determined that a proliferative response of LECs couldbe observed at a concentration of 1000 ng/ml of VEGF-2. Therefore,VEGF-2 samples at a concentration 1000 ng/ml in DMEM were premixed withone of the three different anti-VEGF-2 antibodies (10 μg/ml) and used inthe alamar blue™ assay system to determine the influence on LECproliferation. Controls in the first experiment included VEGF-2 alone(1000 ng/ml), bFGF (10 ng/ml, positive control), IL-2 (irrelevantprotein negative control) and starvation medium (assay negativecontrol). The repeat experiment also included antibody alone (10 μg/ml)as a negative control.

As shown in FIG. 22, VEGF-2 treatment of LECs at a concentration of 1000ng/ml resulted in a proliferative response relative to the negativecontrols, which was consistent with previous proliferative assaysconducted with these cells. Simultaneous treatment of LECs with VEGF-2and monoclonal 13A2 did not augment the proliferative response above thelevel achieved with VEGF-2 alone. However, an enhanced proliferativeresponse was observed with the 13D6 monoclonal and to a lesser degree,with the rabbit polyclonal antibody.

As shown in FIG. 23, the experiment was repeated under more stringentconditions, using 1000 cells/well as an initial cell concentration andincluded stimulation with the antibody alone in order to control forpossible direct effects of the antibodies on the LECs. This experimentdemonstrated augmentation of VEGF-2 mediated proliferation by the 13D6and polyclonal antibodies above the proliferative response observed withVEGF-2 or the antibodies alone. As observed in the previous experiment,the 13A2 antibody did not induce an augmented proliferative response.

These observations suggest that antibody mediated crosslinking of VEGF-2molecules bound to receptors (VEGFR2 or VEGFR3) may induce receptordimerization. Such a process may be used to intensify the signalingresulting from the VEGF-2 binding to its receptors.

Example 10 Mouse Immunization for Monoclonal Antibody Production

Animals are individually housed and received food and water ad libitum.All manipulations are performed using aseptic techniques. Theexperiments are conducted according to the rules and guidelines of HumanGenome Sciences, Inc. Institutional Animal Care and Use Committee andthe Guidelines for the Care and Use of Laboratory Animals.

Dilute concentration of protein in 350 μls of phosphate bufferedsolution (PBS), or other neutral buffer, to a final proteinconcentration of 0.43 mg/ml. With 0.35 mls. Freund's Complete Adjuvant,emulsify the adjuvant and protein solution for a period of ten minutesusing two glass 3 cc syringes and a three way disposable stopcock(Baxter Cat. No. 2C6240). To test emulsion for quality, place 50 μls ofthe emulsion onto the surface of cold water in a beaker. If the emulsiondoes not remain as an intact white droplet, then further mixing isrequired.

Draw all of the emulsion into one syringe, and using a 27 gauge needle,inject mouse subcutaneously with a total of 200 μls of emulsiondistributed among 4-8 sites including axillary and inguinal areas, theback of the neck, and along the back.

Following two to three weeks, repeat the above injection substitutingFreund's Incomplete Adjuvant (as opposed to Freund's Complete Adjuvant).

Following an additional two to three weeks, a third injection is givenas outlined above, making sure to use Freund's Incomplete Adjuvant.

Ten to Fourteen days following the third injection, obtain 100-200 μlsof blood from the mouse by tail vein bleed. Incubate the blood at 37° C.for 60 minutes, and then allow to cool overnight at 4° C. Followingincubation at 4° C., centrifuge the blood for ten minutes. Transfer theserum to a new tube, and test for mouse serum titer. If titer is foundto be low, intraperitoneal (ip) injections can be given at biweeklyintervals. For ip injections, prepare 10-20 μgs protein per mouse in avolume of 200-400 μls of PBS per mouse. Using a 1 cc syringe and a 26gauge needle, inject the solution into the mouse. Do a second tail bleed10-14 days following injection, and retest the mouse serum titer.

Example 11 Mouse Serum Titer ELISA

Coat the ELISA plate with 50 μl/well of purified antigen at 2 μgs/mlPBS. Cover the ELISA plate with parafilm and incubate at 4° C. overnightin a humid chamber. Following incubation, wash the plate four times with200 μl/well of PBS per wash. Block with 3% BSA, 200 μls/well for 60minutes at room temperature. Shake out blocking solution.

Add serum samples in duplicate, 50 μls/well, at dilutions of 10⁻², 10⁻³,10⁻⁴, 10⁻⁵, 10⁻⁶, and 10⁻⁷, diluted in PBS containing 0.1% BSA. Includeblanks of buffer as well as positive and negative control serum at theabove dilutions. Incubate at room temp for 1-2 hours. Wash with PBST(PBS with 0.05% tween), 250 μls/well, four times.

Add 50 μls/well of Biotinylated Anti-Mouse IgG at a concentration of 0.5μg/ml in PBST containing 0.1% BSA and 2% Horse Serum. Incubate at roomtemperature for 30 to 60 minutes. Wash plate four times with PBST.

Add 50 μls/well of ABC reagent (Vector Cat. No. PK-6100) to the plateand incubate at room temperature for 30 minutes. Wash plate six timeswith PBST.

Prepare substrate for ELISA detection by dissolving 1tetramethylbenzidine Dihydrochloride (TMB) tablet (Sigma Cat. No.T-3405) in 5 mls. of ddH₂O. Add 5 mls. of 0.1M Phosphate Citrate Buffer(25.7 mls of 0.2M dibasic sodium phosphate, 24.3 mls of 0.1M citric acidmonohydrate, pH 5.0). Add 2 μls of fresh 30% hydrogen peroxide, vortexand use immediately.

Following incubation and washing of the plate, add 100 μls of substratesolution and incubate at room temperature for approximately 15-30minutes. Stop the reaction by adding 25 μls/well of 2M H₂SO₄, and readthe plate at 450 nm within 30 minutes versus the controls.

Example 12 Fusion Protocolfor Hybridoma Production

One week prior to the fusion step, make P3× growth medium (1× DMEM 0%(Gibco Cat. No. 11965-019), 5-10% Fetal Bovine Serum, 1× L-Glutamine(Biofluids Cat. No. 300), and 1× Sodium Pyruvate (Biofluids Cat. No.333). Thaw a new vial of P3× mouse myeloma cells into 1 well of a 6-welldish (see thawing protocol, infra) and start expanding them in P3×growth medium. If viability is good the next day transfer to a 100 mmdish. Cell density must not exceed 10⁶ cells/ml or greater. Furthermore,membranes of these cells should not look granular. By the day of thefusion procedure there should 6-8 plates at 5-8×10⁵ cells/ml. It is agood idea to test some P3× cells in HAT medium. All cells should be deadwithin around 4 days. If not then P3× cells should be grown in P3×medium containing 15 ug/ml 8-azaguanine to eliminate revertants.

Four days prior to the fusion procedure, the mouse should be immunizedwith an ip injection of approximately 10 μg of high purity protein.

One day before the fusion, split the P3× cells and feed them with freshmedium as needed so that cells will be healthy and growing in log phaseby the next day.

On the day of the fusion procedure, place 50 mls of P3× media, PEGsolution and HAT media (1× DMEM 0%, 20% Fetal Bovine Serum, 4% HybridomaSupplement-BM Condimed HI (Boehringer-Mannheim), 1× L-Glutamine, 1×NEAA, 1× Sodium Pyruvate, 1× HAT (Sigma Cat. No. H0262), 1×0.05M 2ME,and 1× Penicillin-Streptomycin) in a 37° C. water bath. Have availableapproximately 100 ml cold DMEM 0%.

Check all P3× plates for possible contamination and to assess health ofcells. Resuspend cells from 4 plates or flasks and combine in 50 mltubes. Centrifuge at 200× G for 10 min. Aspirate the supernatant.Resuspend each tube with 10 mls DMEM 0% and pool. Count live cells usingtrypan blue viability stain (viability should be greater than 90%). Thetotal number of cells should be 2-4×10⁷ cells. If there are not enoughcells then repeat the process with some more plates. Let cells sit atambient room temperature (ART) until needed in future step.

Prepare hood where spleen will be removed with: 70% EtOH, sterileinstruments including sieve and plunger, 2 petri dishes containing 10 mlDMEM 0% and 15 ml centrifuge tubes (2).

The mouse is sacrificed, and the spleen is then harvested from thecarcass. Place the spleen in the petri dish containing DMEM 0%. Placethe sieve in the other dish containing 10 ml DMEM 0% and cover withplate lid. Transfer the spleen to the sieve using a sterile pair offoreceps, and using the syringes with needles, tease the spleen apart sothat cells spill out into the media. Then, using the other plunger,gently squish the spleen through the sieve. Avoid grinding the spleenorgan tissue through the sieve as this will result in heavy fibroblastgrowth.

Remove the sieve and transfer the spleen cell suspension to a 15 mlcentrifuge tube. Wash remaining cells from the dish with 5 mls DMEM 0%,and add to the tube. Allow the tube to sit for 5 minutes to allow largedebris to settle to the bottom. Then transfer the cell suspension, minusdebris, to the second 15 ml tube. Centrifuge the cells for 10 min. at200× G. Aspirate s/n and resuspend the spleen cells in 5 mls DMEM 0%.Add 5 more mls of DMEM 0%, and transfer the entire volume to a 50 mltube.

Remove 10 μls of the spleen cell suspension, and add to 500 μls ofTrypan blue in order to count lymphocytes. (Note: normally a spleen willconsistently yield 10⁸ lymphocytes).

Fusion

To the 50 ml centrifuge tube containing the spleen cells add sufficientP3× cells to make a lymphocyte to P3× cells ratio of 5:1. (e.g. for 10⁸lymphocytes you will need 2×10⁷ P3× cells). Bring the total volume up to45-50 mls with DMEM 0%, and centrifuge at 200× G for 10 minutes. Preparea transfer hood with a timer, warm PEG, warm P3× media, and a beaker ofwater approximately 38-40° C. Aspirate all of the supernatant from theP3×-lymphocyte pellet and attempt to loosen the pellet by flicking thetube. Place the tube in the small water bath. Keep the fusion tube inthe warm water, and while gently shaking, add 1 ml of PEG dropwise over1 minute. Then, let sit with occasional shaking for 1-2 minutes,following which add 1 ml of P3× media dropwise over 1 minute. Next, add3 mls. of P3× media dropwise over 1 minute, followed by the addition of10 mls. of P3× media dropwise over 1 minute.

Gently add P3× media to make the total volume 45 mls. Allow the tube tosit for 10 minutes, then centrifuge at 200× G for 10 minutes. Aspiratethe supernatant and gently resuspend the pellet in 5 mls or less of HATmedium. Transfer the cell suspension to the bottle containing 400 mls ofHAT medium and swirl to mix. Pour some of the cell suspension into asterile reservoir.

Plant cells in 96 well plates, 200 μls/well, using a 12 channel pipettorwith filtered tips. Place plates in the incubator. Monitor plates everyday for hybridoma growth or contamination. Allow plates to incubate forthree days. The first feeding (medium change) is done by around day 7 byaspirating off approximately half the media in each well using the8-position manifold and replacing it with 100-150 μls/well HT medium.Feeding a week or so before the first screening helps to dilute out anyantibody produced by the unfused lymphocyte cells which have been foundto continue producing antibody after 2 weeks in culture. Many or all ofthe wells will be ready to be sampled for screening within 2 weeks afterthe fusion when the colony or colonies fill more than half the well andthe supernatant has changed color to a orange/yellow.

Example 13 ELISA Screening of Mouse Hybridomas

To screen the mouse hybridomas, coat the ELISA plate (Immulon 2 “U”bottom microtiter plate (Dynatech Cat. No. 011-010-3555)) with 50μls/well of the antigen at 2 μgs/ml PBS. Cover the ELISA plate withplastic seal and incubate at 4° C. overnight. Following incubation, washthe plate four times with 200 μls/well of PBS per wash. Block with 3%BSA, 200 μls/well for 60 minutes at room temperature. Shake out blockingsolution.

Add hybridoma supernatants, 150 μls/well, into a Corning 96 well assayplate, then transfer 50 μls of each supernatant from the Coming assayplate into the ELISA plate. Include blanks of culture medium as well aspositive and negative mouse serum controls. Incubate at room temp for1-2 hours, or overnight at 4° C. Wash with PBST (PBS with 0.05% tween),250 μls/well, four times.

Add 50 μls/well of Biotinylated Anti-Mouse IgG H+L, at a concentrationof 0.5 μg/ml in PBST containing 0.1-0.3% BSA and 1% Horse Serum.Incubate at room temperature for 30 to 60 minutes. Wash plate four timeswith PBST.

Add 50 μls/well of ABC reagent (Vector Cat. No. PK-6100) to the plateand incubate at room temperature for 30 minutes. Wash plate six timeswith PBST.

Prepare substrate for ELISA detection by dissolving 1tetramethylbenzidine Dihydrochloride (TMB) tablet (Sigma Cat. No.T-3405) in 5 mls. of ddH₂O. Add 5 mls. of 0.1M Phosphate Citrate Buffer(25.7 mls of 0.2M dibasic sodium phosphate, 24.3 mls of 0.1M citric acidmonohydrate, pH 5.0). Add 2 μls of fresh 30% hydrogen peroxide, vortexand use immediately.

Following incubation and washing of the plate, add 100 μls of substratesolution and incubate at room temperature for approximately 15-30minutes. Stop the reaction by adding 25 μls/well of 2M H₂SO₄, and readthe plate at 450 nm within 30 minutes versus the controls.

Example 14 Testing Relative Affinity of Monoclonals Derivedfrom CultureSupernatants

A. Determining Antigen Coating Concentration

Make approximately 1 ml of the antigen at a concentration of 4 μgs/ml inPBS. Transfer to a microdilution tube. Place 0.5 ml PBS in each of 9microdilution tubes, then do serial dilutions of ½ by transferring 0.5ml from tube to tube starting from the 4 μgs/ml tube. You will now havetubes containing 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015 and 0.0075μgs/ml. Coat a plate with the above concentrations, 6 wells each, 50μl/well.

Cover and incubate over night at 4° C. Following incubation, wash theplate four times with 200 μls/well of PBS per wash. Block with 3% BSA,200 μls/well for 60 minutes at room temperature. Shake out blockingsolution.

Look at the titer curve of mouse serum which is positive to the antigen.Determine the serum dilution which is just at the top of the titrationcurve. Add the positive mouse serum at this dilution in PBS containing0.1% BSA, 50 μls/well, rows B-D, columns 2-11. Include negative controlserum at the above dilution in rows E-G, columns 2-11. Incubateovernight at 4° C. Following incubation, subtract Negative Control Serumvalues from Positive Control Serum values. Plot mean value (O.D. 450)against antigen concentration on linear scale. Determine antigen coatingconcentration which will give a submaximal O.D. This is the coatingconcentration to use for the relative affinity assay.

B. Determining the Mouse IgG Concentration of the Hybridoma SupernatantSample Using the Boehringer Mannheim Biochemica Kit “Mouse-IgG ELISA”(Cat. No. 1333 151)

Dilute the coating buffer concentrate 1/10 with ddH₂O. Ten to twentymls. will be necessary. Obtain an aliquot of capture antibody. Thaw 3tubes of Post Coating Buffer Concentrate (blocking solution). Twelvewells will be necessary for the standards and 4-6 wells for eachsupernatant to be tested. Calculate the number of mls of diluted CaptureAntibody necessary assuming 50 μls/well of coating volume. DiluteCapture Antibody in the following proportion:

$\frac{25\mspace{14mu} {ul}\mspace{14mu} {Capture}\mspace{14mu} {Ab}}{1\mspace{14mu} {ml}\mspace{14mu} {Coating}\mspace{14mu} {Buff}} = \frac{X\mspace{14mu} {µl}\mspace{14mu} {Capture}\mspace{14mu} {Ab}}{\# \mspace{14mu} {ml}\mspace{14mu} {Coating}\mspace{14mu} {Buff}}$

Coat Nunc plate with the solution and incubate 30 mins at roomtemperature on a shaker. Dilute the concentrated Post Coating Buffer1/10 in ddH₂O. Wash the plate with ELISA wash buffer (0.9% NaCl, 0.1%Tweeen 20), and block with 200 μls/well of Post Coating Buffer (Blocksolution) for 15 minutes at room temperature.

Dilute the IgG standard into Post Coating Buffer (blocking solution) tothe following concentrations: 0.2, 0.1, 0.05, 0.025, 0.0125 and 0.00625μgs/ml in Post Coating Buffer.

Dilute supernatants into blocking buffer to make final concentrations of1/100 and 1/1000. Following the blocking step, wash the plate and add 50μls/well of diluted IgG standards and diluted supernatants in duplicate.Incubate for 30 minutes at room temperature on a shaker.

Dilute the conjugate solution into Post Coating Buffer (block solution)according to the proportion below:

$\frac{{50\mspace{14mu} {µl}\mspace{14mu} {Conjugate}}\mspace{14mu}}{1\mspace{14mu} {ml}\mspace{14mu} {Block}\mspace{14mu} {sol}} = \frac{X\mspace{14mu} {µl}\mspace{14mu} {Conjugate}}{\# \mspace{14mu} {ml}\mspace{14mu} {Block}\mspace{14mu} {sol}}$

Wash the plate and add 50 ul/well of conjugate. Incubate the plate for30 minutes at room temperature on shaker.

Dissolve 1 substrate tablet in 5 mls substrate buffer. Wash the plateand add 50 μls/well of substrate. Incubate 30 minutes at roomtemperature on a shaker and read at 405 nm.

C. Relative Affinity Assay

Coat appropriate ELISA plate(s) overnight at 4° C with the antigenconcentration previously determined. Block plate as above. Make ⅓ serialdilutions into PBS+0.1% BSA of test supernatant.

Add 50 μls/well of the dilutions of the supernatant sample, includingthe undiluted sample, to the ELISA plate(s) in duplicates ortriplicates. The positive control consists of a few wells of thepositive control mouse serum at the same concentration as used fordetermining the antigen coating concentration. The negative controlconsists of a few wells of the dilution buffer. Cover and incubateovernight at 4° C.

Plot the IgG concentration of each supernatant against the mean-value(O.D. 450) on a 4 parameter curve fit. Supernatant curves that are moreto the left are the supernatants with the highest affinities.

Example 15 Ascites Production in Mice

Hybridoma cells should be healthy and in log phase of growth for ascitesproduction. Transfer cells to a 15 ml. tube and count. Determine thevolume which contains 4×10⁶ cells, transfer that volume to a second tubeand centrifuge the cells. Resuspend the pellet in 0.9 mls of HBSS(Hank's Balanced Salt Solution) and transfer to an eppendorf tube.

Fill a 1 cc syringe with the cell suspension and inject mice ip asfollows: 0.2 cc per mouse if the original cell number was 4×10⁶ and 0.3cc if the original cell number was 3×10⁶. When the abdomen is verydistended and slightly taught to the touch, like a balloon, (usually byday 9 or 10 but sometimes as late as day 14) then it is time to “tap themouse”.

A. Tapping:

Hold the mouse in your left hand and use an alcohol pad to wipe off thearea of the abdomen just above the mouse's left hind leg. While holdingthe mouse above an open 15 ml centrifuge tube, insert a 19 gauge needleinto the abdomen. Ascites fluid should immediately begin to drip out ofthe end of the needle into the centrifuge tube An average mouse shouldyield 3-6 mls. of ascites fluid.

B. Processing and Storage of Ascites:

Pool ascitic fluid collected from each mouse in the group (all injectedwith the same hybridoma) and leave at room temperature for 1-2 hours orplace at 37° C. for 15-30 minutes. Then place ascites at 4° C. overnightto allow for clot formation. Centrifuge clotted ascites for 10 minutes.Transfer the liquid ascites to a 50 ml centrifuge tube, and store thetube at −20° C. Subsequent taps may be added to this 50 ml tube. Whenall mice are sacrificed, the pooled ascites can be thawed, respun, andaliquotted for long term storage at −20° C. or −70° C. Ascites should betitered by ELISA.

Example 16 Protocolfor Freezing and Thawing Mouse Hybridoma and MyelomaCells

A. Freezing

Cells to be frozen down should be healthy, in log phase of growth and ata concentration of roughly 5-8×10⁵ cells/ml. Resuspend cells from a 6well plate or flask, transfer to a 15 ml tube and count. Calculate thenumber of total cells and divide by 1-3×10⁶ cells per vial to determinethe number of vials to be frozen down.

Pellet the cells at 200-300×G for 5-10 minutes. Aspirate the supernatantfrom the pellet and resuspend in sufficient cold freeze medium (50% FBS,10% DMSO in DMEM; or Origen DMSO Freeze Medium (IGEN), Fisher Cat. No.IG-50-0715) to achieve the desired number of cells/vial per ml (celldensities should be in a range from 5×10⁵ to 1×10⁷ cells/vial).Immediately transfer the cell suspension to the cryovials, 1 ml pervial, and place on ice. Transfer the vials to a controlled rate freezerand place the freezer at −70° C. for overnight. After 24 hours transferthe vials to a liquid nitrogen tank or −130° C. freezer for long termstorage.

B. Thawing

Add 10 ml cold media (e.g. P3× media) to 15 ml tube. Retrieve cryovialof frozen cells and keep on dry ice until ready to thaw. Thaw cellsquickly in 37° C. water bath. Hold vial during thawing and keep shakinggently until there is just a small bit of ice left in vial. Don't allowthe contents to warm above 4° C. Alcohol off the outside of thecryovial.

Using a sterile pasteur pipette and without touching the edges of thecryovial, transfer the cell suspension in the vial to the 10 mls. ofcold media. Spin at 200-300×G for 5-10 minutes. Aspirate the supernatantand resuspend the pellet in 6 mls. of HT Cloning Media (supra). Transferto 1 well of a 6 well dish. Assess the viability after 24 hours.Viability should not be less than 50%.

Example 17 In Vitro Assay for Angiogenic Protein Activity

The following assay is designed to detect angiogenic protein activity,preferably VEGF-2 activity. For example, a chimeric receptor isgenerated by fusing the nucleotides encoding for the extracellulardomain of the Flt-4 receptor (SEQ ID NO:38) (Galland et al., Genomics 13(2):475-478 (1992), which is herein incorporated by reference in itsentirety), to the nucleotides encoding for the transmembrane domain andintracellular domain of Flk-1 (SEQ ID NO:39) (Davis-Smyth et al., EMBO J15(18):4919-4927 (1996), which is herein incorporated by reference inits entirety). Thus, the chimeric receptor would include amino acids 1to 775 of SEQ ID NO:38, fused to amino acids 765 to 1356 of SEQ IDNO:39, respectively.

Alternatively, the chimeric receptor may be designed as outlined above,but would substitute the transmembrane and intracellular domains of theerythropoietin receptor (EPOR) for the transmembrane and intracellulardomains of the Flk-1 receptor, as discussed in Pacifici et al., JBC269(3): 1571-1574 (1994), which is herein incorporated by reference inits entirety (see specifically FIG. 1).

The resulting DNA encoding for the chimeric receptor is cloned into anappropriate mammalian, baculoviral, or bacterial expression vector, suchas, for example, pC4, pCDNA3, or pA2, as discussed supra. Mammalian hostcells that could be used for expression of the chimeric receptor includeNIH3T3 (supra), or the pre-B cell line BaF3 (Achen et al., PNAS 95(2):548-553 (1998), which is herein incorporated by reference in itsentirety).

To test for activity, the angiogenic protein can be brought into contactwith a cell line expressing the chimeric receptor, or extracts thereof.Then, angiogenic protein binding to the chimeric receptor can bedetected by measuring any resulting signal transduced by the chimericreceptor.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

Additionally, the entire specification, including the Sequence Listingof U.S. application Ser. No. 09/107,997, filed Jun. 30, 1998, and PCTApplication No. US 99/05021 filed Mar. 10, 1999, are hereby incorporatedby reference in their entirety.

1-18. (canceled)
 19. An isolated antibody or fragment thereof that specifically binds to a polypeptide consisting of amino acid residues +9 to +80 of SEQ ID NO: 2, wherein the antibody or fragment enhances a biological activity of VEGF2.
 20. The antibody or fragment of claim 19, wherein said antibody or fragment enhances angiogenesis, neovascularization, vascular permeability, or endothelial cell growth in a mammal.
 21. The antibody or fragment of claim 19, wherein the antibody is assigned ATCC Accession No. PTA-435.
 22. The antibody or fragment of claim 19, wherein the antibody is assigned ATCC Accession No. PTA-199.
 23. The antibody or fragment of claim 19, wherein the antibody is assigned ATCC Accession No. PTA-201.
 24. The antibody or fragment of claim 19 which is a humanized antibody.
 25. The antibody or fragment of claim 19 which is a chimeric antibody.
 26. The antibody or fragment of claim 19 which is a polyclonal antibody.
 27. The antibody or fragment of claim 19 which is a monoclonal antibody.
 28. The antibody or fragment of claim 19 which is a single chain antibody.
 29. The antibody or fragment of claim 19 which is a Fab fragment.
 30. The antibody or fragment of claim 19, comprising a detectable label.
 31. The antibody or fragment of claim 30, wherein the label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent compound; (c) a radioactive compound; and (d) a luminescent compound.
 32. An isolated cell that produces the antibody or fragment of claim
 19. 33. A hybridoma that produces the antibody or fragment of claim
 19. 34. A composition comprising an antibody or fragment of claim 19 and a pharmaceutically acceptable carrier or excipient.
 35. A method of enhancing a biological activity of VEGF2 in a patient comprising administering an isolated antibody or fragment that specifically binds to a polypeptide consisting of amino acid residues +9 to +80 of SEQ ID NO: 2, wherein the antibody or fragment enhances a biological activity of VEGF2.
 36. The method of claim 35, wherein said antibody or fragment enhances angiogenesis, neovascularization, vascular permeability, or endothelial cell growth in a mammal.
 37. A method of enhancing angiogenesis in a patient comprising administering to the patient a therapeutically effective amount of the antibody or fragment of claim
 19. 38. A method of enhancing neovascularization in a patient comprising administering to the patient a therapeutically effective amount of the antibody or fragment of claim
 19. 39. A method of enhancing vascular permeability in a patient comprising administering to the patient a therapeutically effective amount of the antibody or fragment of claim
 19. 40. A method of enhancing endothelial cell growth in a patient comprising administering to the patient a therapeutically effective amount of the antibody or fragment of claim
 19. 41. A method of treating a patient having an injury to or a disorder of an eye comprising administering to the patient a therapeutically effective amount of the antibody or fragment of claim
 19. 42. The method of claim 41, wherein the injury or disorder is selected from the group consisting of: age-related macular degeneration, diabetic retinopathy, peripheral vitreoretinopathies, photic retinopathies, surgery-induced retinopathies, viral retinopathies, ischemic retinopathies, retinal detachment and traumatic retinopathy.
 43. The method of claim 35, wherein the patient is an animal.
 44. The method of claim 43, wherein the animal is a human. 